Plant Cells

00-Titelei 02.07.2001 9:59 Uhr Seite IX (Schwarz/Process Black Bogen) Preface Plants produce more than 30,000 types ...

Author: J. J. Zhong

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00-Titelei 02.07.2001 9:59 Uhr Seite IX

(Schwarz/Process Black Bogen)

Preface

Plants produce more than 30,000 types of chemicals, including pharmaceuticals, pigments and other fine chemicals, which is four times more than those obtained from microbes. Plant cell culture has been receiving great attention as an alternative for the production of valuable plant-derived secondary metabolites, since it has many advantages over whole plant cultivation. However, much more research is required to enhance the culture productivity and reduce the processing costs, which is the key to the commercialization of plant cell culture processes. The recent achievements in related biochemical engineering studies are reviewed in Chapter 1. The effect of gaseous compounds on plant cell behavior has been little studied, and Chapter 2 focuses on these gas concentration effects (including oxygen, carbon dioxide, ethylene and others, such as volatile hormones like methyl jasmonate) on secondary metabolite production by plant cell cultures. Two metabolites of current interest, i.e., the antimalarial artemisinin (known as “qing hao su” in China) that is produced by Artemisia annua (sweet wormwood) and taxanes used for anticancer therapy that are produced by species of Taxus, are taken as examples. Bioprocess integration is another hot topic in plant cell culture technology. Because most of the plant secondary metabolites are toxic to the cells at high concentrations during the culture, removal of the product in situ during the culture can lead to the enhanced productivity. Various integrated bioprocessing techniques are discussed in Chapter 3. To improve the productivity of commercially important compounds in plants or plant cell cultures, or even to produce completely new compounds, metabolic engineering of plant secondary metabolite pathways has opened a new promising perspective. Different strategies used for the genetic modification are discussed in Chapter 4, including single-gene and multiple-gene approaches, as well as the use of regulatory genes for increasing productivity. These approaches are, among others, illustrated with work on the terpenoid indole alkaloid biosynthesis. With the development of genetic engineering of plant cells or organs, a lot of recombinant products can be obtained in cheap plant cell culture media. Production of these high- value products in plant cells is an economically viable alternative to other systems, particularly in cases where the protein must be biologically active. Chapter 5 reviews foreign protein production from genetically modified plant cells, and the implications for future development of this technology are also discussed. Plant micropropagation is another important application of plant cell culture, which is an efficient method of propagating disease-free, genetically uniform

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Preface

and massive amounts of plants in vitro. The prospect of micropropagation through somatic embryogenesis provides a valuable alternative to the traditional propagation system, and the micropropagation of elite hairy roots offers other attractive advantages in the large-scale production of artificial seeds. Large-scale production of somatic embryos and hairy roots in appropriate bioreactors is essential if micropropagation and artificial seed systems are to compete with natural seeds. Chapter 6 identifies the problems related to largescale plant micropropagation via somatic embryogenesis and hairy roots, and the most recent developments in bioreactor design are summarized. Emphasis is given to immobilization technology and computer-aided image analysis employed in the mass micropropagation. As promising materials in plant cell cultures, hairy roots are recently shown to be responsive to physical stimuli such as exposure to light. However, physiological properties of hairy roots caused by environmental conditions have been hardly investigated in engineering aspects. In Chapter 7, the authors have developed the photomixotrophic and photoautotrophic hairy roots of pak-bung (water spinach) from the heterotrophic originals under light conditions. The physiological and morphological properties and growth kinetics of these hairy roots have been characterized. The relationships between growth potential of photoautotrophic hairy roots and energy acquired by photosynthesis in the cells are discussed in terms of maintenance energy. I would like to thank Professor Thomas Scheper, the managing editor of this series, and Dr. Marion Hertel, chemistry editorial of Springer-Verlag, for their strong support. The excellent work and very pleasant cooperation of Mrs. Ulrike Kreusel, desk editor (chemistry) of Springer-Verlag, is greatly appreciated. I am also grateful to the supports from the Cheung Kong Scholars Program of the Ministry of Education of China, the National Natural Science Foundation of China, and the East China University of Science and Technology. ECUST Shanghai February 2001

Jian-Jiang Zhong

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Biochemical Engineering of the Production of Plant-Specific Secondary Metabolites by Cell Suspension Cultures Jian-Jiang Zhong State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China, e-mail: [email protected]

Plant cell culture has recently received much attention as a useful technology for the production of valuable plant-derived secondary metabolites such as paclitaxel and ginseng saponin. The numerous problems that yet bewilder the optimization and scale-up of this process have not been over emphasized. In spite of the great progress recorded in recent years towards the selection, design and optimization of bioreactor hardware, manipulation of environmental factors such as medium components, light irradiation, shear stress and O2 supply needs detailed investigations for each case. Recent advances in plant cell processes, including highdensity suspension cultivation, continuous culture, process monitoring, modeling and control and scale-up, are also reviewed in this chapter. Further developments in bioreactor cultivation processes and in metabolic engineering of plant cells for metabolite production are expected in the near future. Keywords. Plant cell suspension culture, Secondary metabolite production, Bioprocess engineering, Bioreactor optimization, Environmental factors, Medium manipulation, Shear stress, Modeling monitoring and control, High density cell culture, Bioprocess scale-up, Metabolic engineering

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Application of Plant Cell Culture to Production of Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . .

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Design and Optimization of Bioreactor Hardware . . . . . . . . . .

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Manipulation of Culture Environments . . . . . . . . . . . . . . . .

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4.1 4.1.1 4.1.2 4.1.3 4.1.4 4.2 4.3 4.3.1 4.3.2 4.4 4.5

Medium Manipulation . . . . . . . . . . . . . . . . . . . Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . Potassium Ion . . . . . . . . . . . . . . . . . . . . . . . Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . Light Irradiation . . . . . . . . . . . . . . . . . . . . . . Shear Stress . . . . . . . . . . . . . . . . . . . . . . . . . Methods for Evaluating the Shear Effects on Plant Cells Effects of Shear Stress on Plant Cells in a Bioreactor . . Oxygen Supply . . . . . . . . . . . . . . . . . . . . . . . Rheology . . . . . . . . . . . . . . . . . . . . . . . . . .

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Advances in Biochemical Engineering/ Biotechnology, Vol. 72 Managing Editor: Th. Scheper © Springer-Verlag Berlin Heidelberg 2001

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Advances in Bioreactor Cultivation Processes

5.1 5.2 5.3 5.3.1 5.3.2 5.3.3 5.4

High-Density Cell Culture . . . . . . . . . . . Continuous Culture . . . . . . . . . . . . . . Process Monitoring, Modeling and Control . In Situ Monitoring in Shake Flasks . . . . . . On-Line Monitoring of Bioreactor Processes Mathematical Modeling and Process Control Bioprocess Scale-Up . . . . . . . . . . . . . .

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Metabolic Engineering of Plant Cells for Metabolite Production . . 22

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Conclusions and Future Perspectives

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

1 Introduction The history of plant cell culture dates back to the beginning of this century, and since the 1930s a great deal of progress has been achieved. The concept of culturing plant cells includes the culture of plant organs, tissue, cells, protoplast, embryos, and plantlets. The application of plant cell culture has three main aspects: the production of secondary metabolites, micropropagation, and the study of plant cell genetics, physiology, biochemistry and pathology. This chapter reviews the recent advances in the optimization of bioreactor configurations and environmental factors for metabolite production by plant cell suspension culture, new developments in plant cell bioprocesses in bioreactors, and emerging research on metabolic engineering of plant cells.

2 Application of Plant Cell Culture to Production of Secondary Metabolites Plant cell culture has several advantages as a method of producing useful plantspecific metabolites. Plants produce more than 30,000 types of chemicals, including pharmaceuticals, pigments and other fine chemicals, which is four times more than those obtained from microbes. Some of these chemicals are difficult to synthesize chemically, or it is difficult to produce or to increase the amounts produced by microorganisms through genetic engineering. Plant cell culture is not limited by environmental, ecological or climatic conditions and cells can thus proliferate at higher growth rates than whole plants in cultivation. As shown in Table 1, some metabolites in plant cell cultures have been reported to be accumulated with a higher titer compared with those in the parent plants, suggesting that the production of plant-specific metabolites by plant cell culture instead of whole plant cultivation possesses definite merits and poten-

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Biochemical Engineering of the Production of Plant-Specific Secondary Metabolites

Table 1. Product yield from plant cell cultures compared with the parent plants. DW dry

weight Product

Anthocyanin

Anthraquinone Berberine Rosmarinic acid Shikonin

Plant

Vitis sp. Euphorbia milli Perilla frutescens Morinda citrifolia Coptis japonica Thalictrum minor Coleus blumei Lithospermum erythrorhizon

Yield (% DW) Culture

Plant

Yield ratio (culture/ plant)

16 4 24 18 13 10 27.0 14

10 0.3 1.5 2.2 4 0.01 3.0 1.5

1.6 13.3 16 8 3.3 1000 9 9.3

tial. Because plant cell culture is usually of low productivity, it can only be economically viable in the production of high-value added metabolites [1]. Generally, one main problem in the application of plant cell culture technology to secondary metabolite production is a lack of basic knowledge concerning biosynthetic routes and the mechanisms regulating the metabolite accumulation. Recently, there have been some reports addressing this important issue in plant cell cultures through elicitation, cell line modification by traditional and genetic engineering approaches, as well as biochemical study. Elicitation is effective in enhancing metabolite synthesis in some cases, such as in production of paclitaxel by Taxus cell suspension cultures [2] and tropane alkaloid production by suspension cultures of Datura stramonium [3]. Increasing the activity of metabolic pathways by elicitation, in conjunction with end-product removal and accumulation in an extractive phase, has proven to be a very successful strategy for increasing metabolite productivity [4]. For example, two-phase operation with elicitation-enhanced alkaloid production in cell suspension cultures of Escherichia californica [5, 6]. Cell line selection is one of the traditional and effective approaches to enhancing metabolite accumulation, and biochemical studies provide the fundamental information for the intentional regulation of secondary metabolism in plant cells. In a carrot suspension culture regulated by 2,4-dichlorophenoxyacetic acid, Ozeki et al. [7] found that there was a correlation between anthocyanin synthesis and morphological differentiation for somatic embryogenesis; they also demonstrated the induction and repression of phenylalanine ammonia lyase (PAL) and chalcone synthase correlated with formation of the respective mRNAs. Two biosynthetic enzymes, i.e., PAL and 3-hydroxymethylglutaryl-CoA reductase, were also related with shikonin formation in Lithospermum erythrorhizon cultures [8]. Although plant cell culture has been demonstrated to be a useful method for the production of valuable secondary metabolites, many problems arise during bioprocess scale-up (Table 2). At present, there are only a few industrial processes in operation using plant cell cultures, which include shikonin, ginseng

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Table 2. Problems in plant cell cultures

Biological

Operational

Slow growth rate Physiological heterogeneity Genetic instability Low metabolite content Product secretion

Wall adhesion Light requirement Mixing Shear sensitivity Aseptic condition

and paclitaxel production. Whether or not more products produced in this way will reach the market is strongly contingent on the economics of the process, which in turn is heavily dependent on the productivity of the culture. As mentioned above, selection of cell lines with suitable genetic, biochemical and physiological characteristics is an important point. Optimization of bioreactor configurations and environmental conditions is also definitely necessary to realize commercial production of useful metabolites by plant cells. In the following sections, the environmental factors emphasized are medium components, light, shear, O2, and rheology; the effect of gas concentration on secondary metabolite production by plant cell cultures has been reviewed by Linden et al. [9]. The developments in plant cell bioprocesses including highdensity cell cultivations, continuous culture, process monitoring, modeling and control and process scale-up are focused; process integration for plant cell cultures has been reviewed by Choi et al. [10].

3 Design and Optimization of Bioreactor Hardware Most of the bioreactors used to grow plant cells are usually directly derived from microbial fermenters. The choice and design of the most suitable reactor is determined by many factors, such as shear environment, O2 transfer capacity, mixing mechanism, foaming problem [11], capital investment [12] and the need for aseptic conditions, related to the type of plant cells used and the purpose of the experiment [1]. Understanding how to promote better cell culture through reactor modification, e.g., impeller designs that produce reduced shear, and the efficient introduction of light, is a major challenge [13]. Until now, bioreactors of various types have been developed. These include loop-fluidized bed [14], spin filter, continuously stirred turbine, hollow fiber, stirred tank, airlift, rotating drum, and photo bioreactors [1]. Bioreactor modifications include the substitution of a marine impeller in place of a flat-bladed turbine, and the use of a single, large, flat paddle or blade, and a newly designed membrane stirrer for bubble-free aeration [13, 15–18]. Kim et al. [19] developed a hybrid reactor with a cell-lift impeller and a sintered stainless steel sparger for Thalictrum rugosum cell cultures, and cell densities of up to 31 g l–1 were obtained by perfusion without any problems with mixing or loss of cell viability; the specific berberine productivity was comparable to that in shake flasks. Su and Humphrey [20] conducted a perfusion cultivation in a stirred tank bio-

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reactor integrated with an internal cross-flow filter which provided O2 without bubbles; a cell density of 26 g dry wt l–1 and a rosmarinic acid productivity of 94 mg l–1 d–1 were achieved. In Catharanthus roseus cell cultures, a double helical-ribbon impeller reactor with a working volume of 11 l was successfully developed for high-density cultivation [21]. Yokoi et al. [22] also developed a new type of stirred reactor, called a Maxblend fermentor, for high-density cultivation of plant cells, and they demonstrated its usefulness in cultivations of rice and shear-sensitive Catharanthus roseus cells. In suspension cell cultures of Perilla frutescens for anthocyanin pigment production, an agitated bioreactor with a modified sparger and with internal light irradiation was developed, and the red pigment accumulation in the bioreactor was greatly enhanced compared with that obtained in a conventional reactor [23]. Recently, based on the principles of a centrifugal pump, a new centrifugal impeller bioreactor (CIB) (Fig. 1) has been designed for shear-sensitive biological systems by installing a centrifugal-pump-like impeller in a conventional stirred vessel [24, 25]. The fluid circulation, mixing, and liquid velocity profiles in the new reactor (5-l) were assessed as functions of the principal impeller design and bioreactor operating parameters. The performances of the CIB were compared with those of a widely used cell-lift bioreactor. The effects of the impeller configuration, aeration rate, and agitation speed on the oxygen transfer coefficient were also studied in the CIB. It was understood that the CIB showed higher liquid lift capacity, shorter mixing time, lower shear stress, and favorable oxygen transfer rate compared with the cell-lift counterpart. The

Fig. 1. Schematic diagram of a centrifugal impeller bioreactor (5 l). 1 Magnetic stirring de-

vice; 2 gas in; 3 head plate; 4 agitator shaft; 5 measurements of the liquid velocity profiles of the discharge flow were performed in the vertical direction across the width of the blades (the vertical dashed line) and at various radial distances from the impeller tip; 6 sintered stainless sparger; 7 centrifugal blade; 8 draft tube; 9 DO probe; 10 centrifugal rotating pan

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CIB could have a promising future in its application in shear sensitive cell cultures [26]. Furthermore, the CIB was applied to cell suspension cultures of Taxus chinensis [27] and Panax notoginseng [28]. It was shown that, although the specific growth rate of Taxus chinensis cells in both the CIB and the cell-lift bioreactor was almost the same, the cells cultured in the CIB had shorter lag phase and less cell adhesion to the reactor wall compared with that in the cell-lift reactor [27]. The accumulation of anticancer diterpene paclitaxel was also higher in the CIB (data not shown). For high-density suspension cultivation of Panax notoginseng cells [28], the maximum cell density reached 28.9, 26.0 and 22.7 g l–1 (by dry weight) in a shake flask (SF), the CIB and a turbine reactor (TR), respectively; their corresponding biomass productivity was 1103, 900 and 822 mg l–1 d–1. The total production of ginseng saponin reached about 0.92, 0.80, and 0.49 g l–1 in the SF, CIB and TR, respectively; their corresponding saponin productivity was 34, 29 and 21 mg l–1 d–1. It can be concluded that, from the viewpoint of biomass production and saponin accumulation, the CIB was better than the TR, and the flask culture results can be well reproduced in the CIB. Here, it can be seen that during the last decade research on the design of plant cell bioreactors has witnessed a boom and reached maturity. A future trend in this area is to combine bioreactor type with operational conditions for specific cell lines of characteristic morphology, physiology and metabolism, in order to optimize the processes for secondary metabolite production.

4 Manipulation of Culture Environments 4.1 Medium Manipulation

Plant cell culture medium includes phytohormone, inorganic and organic components. The effects of the medium employed on various processes have been reported, e.g., plant hormone effect in suspension cultures of Panax quinquefolium [29], effects of calcium and phosphate in the cultivation of Coffea arabica suspended cells [30], phosphate effects on saponin production in suspension cultures of Panax ginseng, Panax notoginseng, and Panax quinquefolium [31, 32], and the role of glucose in ajmalicine production by Catharanthus roseus cell cultures [33]. It is evident that medium manipulation is a very powerful way of enhancing the culture efficiency of plant cell cultures. A few more detailed examples, mainly from our laboratory regarding sucrose, nitrogen, potassium ion and medium feed, are illustrated below. 4.1.1 Sucrose

Carbon source was found to be a significant factor in plant cell metabolism [34–39], which affected the accumulation of alkaloids by suspension cultures of Holarrhena antidysenterica [36], of anthocyanins by Vitis vinifera cell suspen-

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sions [37], and of shikonin by Lithospermum erythrorhizon cell cultures [38]. A relatively higher concentration of sucrose was reported to be favorable to the rosmarinic acid production [34, 35]. In suspension cultures of ginseng cells (Panax spp.) for simultaneous production of ginseng saponin and ginseng polysaccharide, which both possess antitumor and immunological activities, manipulation of medium sucrose was demonstrated to be very effective for improvement of culture productivity [39]. The effect of initial sucrose concentration (i.e., 20, 30, 40 and 60 g l–1) was investigated in cell cultures of Panax notoginseng. The final dry cell weight was increased with an increase in initial sucrose concentration from 20 to 40 g l–1, but an even higher sucrose concentration of 60 g l–1 seemed to repress the cell growth. A high sugar level was favorable to the synthesis of ginseng saponin, which may be due to the high osmotic pressure and reduced nutrient uptake (especially nitrate) under the conditions [39]. The content of ginseng polysaccharide was not apparently affected by initial sucrose levels. The maximum production of ginseng saponin and polysaccharide was achieved at an initial sucrose concentration of 40 g l–1. 4.1.2 Nitrogen

Nitrogen source is also very important for plant cell metabolite formation, as reported in suspension cultures of Holarrhena antidysenterica for accumulation of alkaloids [36], in cell suspensions of Vitis vinifera for anthocyanin formation [37], and in shikonin production by Lithospermum erythrorhizon cell cultures [38]. The effects of nitrogen (N) source on the kinetics of cell growth, major nutrient consumption, and production of ginseng saponin and polysaccharide in suspension cultures of Panax ginseng cells have been studied [40]. The ratio of NO3–/NH+4 and initial total medium N were varied. Cell growth was better at 60 mM N and higher ratios of NO3–/NH4+. With nitrate alone or a mixture of nitrate and ammonium (at a ratio of 2:1) provided as N source, 10 mM N was found to be a crucial point for the cell mass accumulation. Nitrate was a favorable N source for ginseng cell growth, and a cell growth rate of 0.11 per day and a dry cell density of 13 g l–1 were obtained at 10 mM of nitrate (sole N source). The results also indicate that the polysaccharide content was not much affected by a variation of the N source, while its maximum production of 1.19 g l–1 was achieved at NO–3/NH4+ of 60:0 under 60 mM of total N (i.e., sole nitrate). The saponin production was relatively higher with an initial N concentration of 5–20 mM (with nitrate alone or at an NO3–/NH4+ ratio of 2:1). 4.1.3 Potassium Ion

Study of the potassium ion has been neglected [41] compared with other mineral ions like Cu2+ [42, 43], although it plays important biochemical and biophysical roles in plant cells. K+ serves as a major contributor to osmotic poten-

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Table 3. Effects of initial K+ concentration on the maximum content, production, productivity (Pv) and yield (against sugar) of ginseng saponin (S) and polysaccharide (P) in suspension cultures of Panax ginseng in shake flasks

Initial K+ (nM)

Content (mg g–1)

Production (g l–1)

Pv (mg l–1 d–1)

Yield (%)

S

P

S

P

S

P

S

P

0 5 10 20 30 40 60

50 54 54 48 63 71 76

100 95 98 91 90 91 90

0.35 0.44 0.45 0.45 0.62 0.70 0.73

0.72 0.78 0.81 0.88 0.89 0.90 0.84

6.5 7.3 7.6 7.6 14 14.6 15.4

14.2 15.8 16.6 18.6 19 23 21

1.0 1.3 1.3 1.4 1.9 2.3 2.2

2.1 2.3 2.7 2.8 2.5 3.0 2.8

tial, a specific requirement for protein synthesis, and an activator for particular enzyme systems. However, almost all previous studies have concentrated on cultivated plants, and there is only one early report related to K+ effect on plant suspension cells [44]. In addition, it should be noted that during an investigation on the effects of nitrogen sources in plant cell cultures, in reality the K+ amount is significantly altered because KNO3 is a major medium component in conventional media. Thus, in such a case, the K+ effect should also be clarified. Table 3 shows the effects of initial K+ concentrations within the range of 0–60 mM on the kinetics of cell growth, nutrient metabolism, and production of ginseng saponin and polysaccharide in suspension cultures of Panax ginseng cells, which were studied by varying KNO3 and NaNO3 concentrations at 60 mM of total nitrate (as the sole nitrogen source) [41]. It is understood that a higher K+ concentration sustained a longer cell growth stage, and the changes in specific oxygen uptake rate (SOUR) of the cultured cells corresponded to the cell growth pattern with the maximum SOUR occurring before the growth peak. More soluble sugar was stored within the cells under K+ deficiency. A curvilinear relationship between initial K+ concentration and the active biomass accumulation (which was the total cell mass minus intracellular soluble sugars) was found, and the critical K+ concentration was determined to be 20 mM. Furthermore, the results indicate that K+ had little effect on the specific production (i.e., content) of ginseng polysaccharide; however, with an increase of initial K+ within 0–20 mM, the total production of polysaccharide was increased gradually due to the increase in cell mass, and it leveled off at K+ over 20 mM. On the other hand, the specific saponin production was remarkably enhanced with an increase of initial K+ within 20–60 mM, and the maximum saponin production was achieved at an initial K+ concentration of 60 mM. 4.1.4 Feeding

Medium feeding is an effective way to improve the plant cell density and production of secondary metabolites [45–47]. For example, Wang et al. [46] de-

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Table 4. Effects of feeding of carbon and nitrogen sources on cell growth and the content (C),

production (Pn) and productivity (Pv) of ginseng saponin and polysaccharide. The cell culture in a modified MS medium was taken as a control. Cases 1, 2 and 3: Sucrose was fed beginning from day 15, 18 and 20, while its level was kept below 20, 10 and 20 g l–1, respectively. Cases 4, 5 and 6: Feeding of 20 mM nitrate, 10 mM nitrate plus 10 mM ammonium, and 20 mM ammonium on day 15, respectively, where sugar addition was the same as in case 1 Case

Control 1 2 3 4 5 6

Dry cells (g l–1)

24.1 35.1 30.6 31.3 28.9 30.3 26.9

Saponin

Polysaccharide

C (mg g–1)

Pn (g l–1)

Pv (g l–1 d–1)

C (mg g–1)

Pn (g l–1)

Pv (g l–1 d–1)

56.8 44.8 36.1 33.6 47.1 29.0 22.7

1.37 1.57 1.10 1.05 1.36 0.88 0.61

0.061 0.055 0.037 0.035 0.047 0.028 0.018

14.2 14.9 14.7 14.6 13.2 11.4 10.2

3.42 5.22 4.50 4.57 3.81 3.44 2.74

0.152 0.186 0.158 0.161 0.132 0.118 0.091

monstrated a conspicuous increase in taxane diterpene production by sucrose feeding. As shown by Fett-Neto et al. [47], paclitaxel yield was enhanced by aromatic carboxylic acid and amino acid feeding to cell cultures of Taxus cuspidata. Table 4 shows the effects of carbon and nitrogen source feeding on the cell cultures of Panax notoginseng [45]. In case 1, the highest dry cell weight of 35.1 g l–1 was obtained, and, as observed, the cell sedimentation volume reached nearly 100%. The cell density reached may be the upper limit of this cell line. The highest production of ginseng saponin and polysaccharide was 1.57 and 5.22 g l–1, respectively. The productivities of ginseng saponin and polysaccharide obtained were 2.8- and 3.4-fold higher than those reached in a conventional batch culture (i.e., in standard MS medium), respectively. The saponin content in case 1 was higher than that in case 2 (at a relatively low sugar level); this may be due to the fact that a relatively higher sugar concentration was favorable to ginseng saponin synthesis. Case 3 (with too late feeding) was also lower in saponin formation compared with case 1, which was probably due to the decline in cell activity. In the cases of 4, 5 and 6, feeding of nitrogen source during the later part of cultivation seemed to have a small negative effect on the cell growth, although sugar was fed at the same time as in case 1. This means that the initial nitrogen amount was enough for the high-density cell culture and the phenomenon of substrate inhibition may have occurred during cultivation. Based on the above results, the best approach for high-density cultivation of Panax notoginseng cells is to start the sugar feeding at the later exponential phase (i.e., day 15), and to keep the medium level below 20 g l–1 by intermittent addition. This strategy has also been successfully applied to bioreactor cultivation of Panax notoginseng cells.

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4.2 Light Irradiation

The spectral quality, intensity and period of light irradiation may affect plant cell cultures in one aspect or another [48]. Various research groups have demonstrated the stimulatory effect of light irradiation on the formation of compounds such as anthocyanins, vindoline, catharanthine, and caffeine in cell suspension cultures [48, 49]. Zhong et al. [48] investigated the quantitative effect of light irradiation intensity on anthocyanin formation by Perilla frutescens cell cultures, and found that the control of light intensity at 27.2 W/cm2 was favorable to the red pigment production in a bioreactor. 4.3 Shear Stress

In suspension culture systems, the cells are subjected to hydrodynamic forces arising from mechanical agitation and sparging aeration. The effect of shear on biological cells has been investigated in various studies. Plant cells are perhaps not as sensitive as animal cells to hydrodynamic forces but are in general more sensitive than microbial cells because they have a large volume (with a size even larger than animal cells) and rigid (inflexible) cell wall. They also tend to form even larger aggregates up to a few mm in diameter [50, 51]. The cell morphology and aggregate size are very dependent on the hydrodynamic shear forces in the surrounding fluid. Therefore, plant cells are also considered to be shearsensitive. In addition, plant cells in suspension culture tend to adhere to the walls of the culture vessels and accumulate at the headspace. At high cell densities, the biomass volume can take up 50–90% of the culture volume because of the high water content of plant cells. All these characteristics add complexity to operating large-scale bioreactors for suspension culture of plant cells [50, 51]. The shear-sensitivity of plant cells has been observed in a number of cases [17, 52–58]. For example, Takeda et al. [58] reported decreases in respiration rate, ATP content and membrane integrity of both eucalyptus and safflower cells under hydrodynamic stress in a stirred-tank reactor. 4.3.1 Methods for Evaluating the Shear Effects on Plant Cells

Quantitative investigations of shear effects on plant cells have been carried out in well-defined flow and shear fields using special flow and shearing devices such as Couette-type apparatus, recirculating flow capillary, submerged jet, or in well-characterized bioreactors. To date various methods have been developed and employed for the assessment of shear-related effects in plant cell suspension cultures (Table 5) [59, 60]. The evaluation of the shear effects on plant cells can be carried out by two approaches: a short-term shearing experiment to observe such parameters as viability and cell lysis, or a long-term bioreactor cultivation to determine the cell growth rate and metabolite productivity. Both cases are demonstrated below by examining our work on Perilla frutescens cells.

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Table 5. Methods for assessing shear-related effects in plant cell suspension cultures [59, 60].

TTC Triphenyltetrazolium chloride System response

Parameter measured

Reduction in viability

Growth rate Regrowth potential Membrane integrity Dye exclusion Dual isotope labelling Dielectric permitivity Release of intracellular components pH variation Protein release Total organic carbon Secondary metabolite release Change in metabolism Oxygen uptake rate Respiration activity (TTC reduction) ATP concentration Metabolite productivity Cell wall composition Changes in morphology and/or aggregation patterns Aggregate size/shape Growth and lysis kinetics Expansion index Esterase release

4.3.2 Effects of Shear Stress on Plant Cells in a Bioreactor

Plant cells cultivated in a bioreactor may be affected by shear stress created by agitation and/or aeration during a long-term exposure to even a low-shear environment. Here, a quantitative investigation of the influence of hydrodynamic shear on the cell growth and anthocyanin pigment production is demonstrated by using a plant cell bioreactor with marine impellers of larger (85 mm) and smaller (65 mm) diameter (Fig. 2) [61]. Figures 2A and 2B show the effect of the average and maximum (represented by the impeller tip speed) shear rates on the specific growth rate of Perilla frutescens cells in a bioreactor. The specific growth rate was apparently reduced at an average shear rate over 30 s–1 or at an impeller tip speed of over 8 dm s–1. Figure 2C and 2D show the influence of shear on the maximum cell concentration of the cell cultures. The maximum cell concentration was relatively higher at an average shear rate of 20–30 s–1 or an impeller tip speed of 5–8 dm s–1. The cell density was reduced at an average shear rate over 30 s–1 or at an impeller tip speed over 8 dm s–1. The effect of shear stress on the pigment content, i.e., its specific production, of the Perilla frutescens cells in the reactor is shown in Figs. 3A and 3B. The anthocyanin content was relatively high at an average shear rate below 30 s–1 or an impeller tip speed below 8 dm s–1. At higher shear rates, the pigment content of the cultured cells showed an obvious decrease. The effects of shear on the total production and the volumetric productivity of anthocyanin in the bioreactor were also similar (data not shown). The pigment production and pro-

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Fig. 2. Influences of shear on specific growth rate and maximum cell concentration of Perilla frutescens cells in a plant cell reactor with a marine impeller. Circles larger impeller (85 mm diam.); triangles smaller impeller (65 mm diam.)

ductivity were relatively higher at an average shear rate between 20–30 s–1 or an impeller tip speed between 5–8 dm s–1, and they decreased at higher shear rates. The effect of shear rate on the yield coefficient of anthocyanin against sucrose in the cell cultures is shown in Figs. 3C and 3D. A relatively higher product yield was observed at an average shear rate between 20–30 s–1 or an impeller tip speed between 5–8 dm s–1. Such information about shear effects in plant cell suspension cultures as given above is useful for bioreactor design and operation as well as the optimization of bioreactor environments for plant cell cultures. It may also be beneficial to bioreactor scale-up and high-density cultivation of plant cells for production of useful plant-specific chemicals (pharmaceuticals). In addition, because different cell suspensions can show different degrees of cell sensitivity to shear stress, and shear affects culture viability, cell lysis, and even metabolite secretion, as demonstrated in various cases like cell cultures of tobacco, Catharanthus roseus and Perilla frutescens [17, 54, 61], detailed studies are required for individual cases.

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Fig. 3. Effects of shear on anthocyanin content and product yield in bioreactor cultures of Perilla frutescens cells. The symbols are the same as in Fig. 2

4.4 Oxygen Supply

O2 supply affects both growth and metabolite production in a number of plant cell cultures including Perilla frutescens [23] and Catharanthus roseus [62]. In flask cultures of Thalictrum minus cells for berberine production, berberineproducing cells were observed to take up twice as much O2 as nonproducing cells [63]. Gao and Lee [64] also demonstrated that an increase in the O2 supply improved the specific O2 uptake rate, the formation of a foreign protein (b-glucuronidase), and secondary metabolite (phenolics) production in flask and bioreactor cultivations of tobacco cells. Recently, Schlatmann et al. [65, 66] reported the effects of oxygen and dissolved oxygen concentration on ajmalicine production and related enzyme activities in high-density cultures of Catharanthus roseus. For anthocyanin production by cell cultivation of Perilla frutescens in an agitated bioreactor, at an aeration rate of 0.1 vvm using a sintered sparger, its accumulation was poor, showing almost the same result as that at 0.2 vvm using a ring sparger, when the other cultivation conditions were maintained the same [23]. However, when the aeration rate was increased to 0.2 vvm with the sinter-

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ed sparger being used, the product formation increased to almost threefold (i.e., 1.65 g l–1). This result suggests that the oxygen supply conditions had a conspicuous effect on secondary metabolite production. 4.5 Rheology

The rheology of plant cell suspension cultures is important for the development and improvement of cell cultivation processes. Knowledge of the rheology of plant cell cultures may suggest solutions to various problems because culture viscosity, mixing, mass transfer, shear stress and cell growth, as well as metabolite production, all interact in bioreactor cultivation [67–70]. Cell cultures of Perilla frutescens were found to exhibit Bingham plastic fluid characteristics, and the size of the individual cells, not the cell aggregates, affected the rheological characteristics [67]. Ballica et al. [71] studied the rheological properties and determined the yield stress value of Datura stramonium cell suspensions. Curtis and Emery [72] demonstrated the rheological characteristics of 10 different plant cell suspension cultures, and claimed that most plant cell suspensions displayed Newtonian behavior at moderate cell densities, and nonNewtonian behavior was a result of cellular elongation observed in only a few cell lines. With an increase in apparent viscosity of cell suspension, a significant decrease in KLa was observed [73]. The results of oxygen supply effects on anthocyanin production by Perilla frutescens [23] imply that the efficiency of the production process may be reduced with an increase in the culture viscosity during cultivation. In addition, the problem of oxygen supply may become more serious in high-density bioreactor cultivations where poor mixing and heavy sedimentation can occur, thus improvement in metabolite production may become difficult. Recently, there has been an interesting report about the regulation of broth viscosity and O2 transfer coefficient by osmotic pressure in cell suspensions of Panax notoginseng [74], while a relatively higher osmotic pressure was found favorable to the saponin accumulation by the cells [75].

5 Advances in Bioreactor Cultivation Processes 5.1 High-Density Cell Culture

Until now, the limited commercial application of plant cell cultures can be attributed to matters of production cost, while economic feasibility studies are unanimous in acknowledging productivity as a limiting factor. Due to the fact that productivity depends on product yield, the organism growth rate and prevailing biomass levels, high-density cell culture systems offer the advantage of production of metabolites in a compact cultivation vessel with high volumetric production rate (i.e., productivity), especially for intracellular products. For example, in Perilla frutescens cell cultures for anthocyanin production [76] and in

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Fig. 4. Schematic diagram of a concentric-tube airlift bioreactor (with working volume of 1.0 l). 1 Reactor column; 2 inner draft tube (with its height of 15 cm); 3 sparger; B bottom clearance (2.5 cm); d diameter of draft tube (4.5 cm); D reactor diameter (7.0 cm); H column height (40 cm)

cultivation of Panax notoginseng cells in stirred bioreactors for the production of ginseng biomass and ginseng saponin [28], high-density cultivation has been successfully demonstrated. A feeding strategy was studied in an airlift bioreactor (Fig. 4) in order to enhance the productivities of both ginseng saponin and ginseng polysaccharide on a reactor scale. It was found that sucrose should be fed before the specific oxygen uptake rate (SOUR) experiences a sharp decrease (at day 13) during the cultivation, i.e., the SOUR drop rate should not be larger than 0.5 mg O2 (g DW)–1 h–1 d–1. The added sugar could not be efficiently used for cell growth and metabolite synthesis if it was fed when the SOUR of the cells had already undergone a sharp drop and was very low (Table 6). Figure 5 shows the kinetics of cell growth and sugar consumption in the airlift fed-batch cultivation (AFB). It indicates that sugar feeding did promote the final cell density, and a very high cell concentration of 29.7 g DW l–1 was successfully achieved on day 17. A very high biomass productivity of 1518 mg DW l–1 d–1 was also obtained in this case. The cell density was comparable to a previous record in flask cultures [45], while the productivity obtained here was much higher than that case, where it was 1188 mg DW l–1 d–1 [75]. In high-density cultures of carrot cells in a 1-l airlift reactor, the highest cell density obtained was 15.2 g l–1 (1.09 ¥ 107 cells ml–1) [77]. In the case of Catharanthus roseus

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Table 6. Comparison of production and productivity of cell mass (by DW), saponin and polysaccharide under different feeding strategies

Feeding time

Day 13

SOUR (mg O2 g–1 (DW) h–1) Total production (g l–1)

7.9 ± 0.5

Productivity (mg l–1 · d–1) a

Dry cells Saponin Polysaccharide Dry cells Saponin Polysaccharide

29.7 ± 1.3 (17) a 2.1 ± 0.2 3.0 ± 0.3 1518 ± 82 106 ± 14 158 ± 19

Day 15 4.2 ± 0.6 26.7 ± 0.8 (17)a 1.7 ± 0.1 2.5 ± 0.2 1347 ± 53 88 ± 10 125 ± 14

The number in parentheses means the time (day) when the maximum cell dry weight was obtained, and the maximum metabolites’ production was also reached on the same day. The data are expressed as an average with standard deviation, which was calculated from three samples.

suspension cultures, the maximum biomass accumulation obtained in an airlift reactor was 23.4 g l–1, although a higher cell density over 30 g l–1 was achieved in a shake flask control [78]. A fluctuation in saponin content was seen in the AFB (Fig. 5B). The final production of ginseng saponin in the AFB reached about 2.1 g l–1, and its productivity was 106 mg l–1 d–1. Both the production titer and volumetric productivity were much higher than those previously reported on a flask scale [45]. In the AFB, the dynamic change in polysaccharide content ranged from 7.3 mg (100 mg DW)–1 to 10.2 mg (100 mg DW)–1 (Fig. 5C). Generally, it increased with an increase in cell mass from day 5. Because of the high cell density, the total production of polysaccharide in the bioreactor was up to 3.03 g l–1 on day 17, and its productivity reached 158 mg l–1 d–1. 5.2 Continuous Culture

Theoretically speaking, continuous culture is most promising for obtaining a high fermentation productivity. Seki et al. [79, 80] and Phisalaphong and Linden [81] demonstrated the enhancement of paclitaxel production by continuous cell culture of Taxus cuspidata and by using a semicontinuous culture of Taxus canadensis with total cell recycle, respectively. However, due to the practical problems of cell line stability and long-term sterile operation of bioreactors, such work is still limited in the laboratory. On the other hand, as shown by Van Gulik et al. [82], a chemostat culture technique is useful to obtain reliable data on the stoichiometry of the growth of plant cells in a bioreactor. Several other groups have also studied growth kinetics, stoichiometry, and modeling of the growth of suspension-cultured plant cells by using semicontinuous or fed-batch cultures to achieve steady-state growth. In addition, Westgate et al. [83] presented fed-batch cultivation kinetics for continuous approximation in Cephalotaxus harringtonia cultures.

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Fig. 5. Time profiles of A fresh and dry cell weights, medium sugar and SOUR, B the content and total production of ginseng saponin and C polysaccharide in fed-batch cultivation of Panax notoginseng cells in 1-l airlift reactors. The arrow in A indicates the sucrose feeding point. The error bars in the figure indicate the standard deviations for the samples of three identical reactors. Symbols: A Dark circles FW; open circles DW; open triangles, medium sugar; dark triangles, SOUR. B Dark circles Saponin production; open circles saponin content. C Dark circles Polysaccharide production; open circles polysaccharide content

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5.3 Process Monitoring, Modeling and Control

For a better understanding of the dynamic changes in the physiological state of cells during cultivation, as well as for control and optimization of bioprocesses, monitoring of physiological variables is very important. For example, the shear damaging effects could be well understood by monitoring the O2 uptake rates of suspended strawberry cells [84]. In spite of a pressing need for better monitoring and control in the optimization of plant cell bioprocesses, few studies have been published in this area. The monitoring parameters most frequently reported in plant cell cultures are the cell concentration, the NAD(P)H concentration [85], and the O2/CO2 concentrations in reactor inlet and outlet gases [86]. The monitoring of cell concentration is one of the most attractive areas of research [87], and in plant cell suspension culture it has been performed by conductometry [88, 89], osmotic pressure measurement [90], the dielectric measurement [91, 92] and turbidimetry [93, 94], and it can also be estimated by a neural-network approach [95]. Furthermore, in pigment production by cell suspension cultures, Smith et al. [96] demonstrated a nondestructive machine vision analysis of pigment-producing cells, and Miyanaga et al. [97] analyzed pigment accumulation heterogeneity using an image-processing system. Recently, Yanpaisan et al. [98] used flow cytometry to monitor cell cycle activity and population on dynamics in heterogeneous plant cell suspensions. Such work provides an opportunity for finetuning bioprocesses based on cell-level phenomena. 5.3.1 In Situ Monitoring in Shake Flasks

In plant cell cultures, shake flask culture is an indispensable stage of cultivation. Investigations in a shake flask are very essential and critical to bioprocess scaleup and optimization. We have developed a simple and convenient technique based on the principle of the Warburg manometric method to measure O2 uptake rate (OUR) and CO2 evolution rate (CER) of suspended cells in a shake flask culture. This technique has been successfully applied to suspension cultures of Panax notoginseng cells, and some important bioprocess parameters, such as OUR, CER, respiratory quotient (RQ), SOUR and specific CER (SCER), were quantitatively obtained [99]. As long as the environment temperature is strictly controlled to within an error of 0.1 °C, the measuring system is accurate and reproducible, is easy to operate, is economical, and is also able to treat many samples simultaneously. 5.3.2 On-Line Monitoring of Bioreactor Processes

A computer-aided on-line monitoring system was established and applied for plant cell bioprocesses, with Perilla frutescens as a model cell [86]. The cell concentration was successfully measured on-line and in situ with a laser turbidi-

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Fig. 6. Dynamic behaviors in total anthocyanin (TA), anthocyanin content (AC), oxygen uptake rate (OUR) and specific oxygen uptake rate (SOUR) in a bioreactor. Triangles 150 rpm; circles 300 rpm; diamonds 400 rpm agitation speed

meter (at 780 nm) [94]. Using this system, a detailed analysis of the response of the cells’ physiological and metabolic aspects such as cell growth and respiratory activity under different agitation speeds was conducted. The system was useful for the selection of some informative process variables and for the identification of the physiological states of plant cells during bioreactor cultivations. The process variables, such as RQ, pH, and SCER, could be used for the identification of the growth phase of the cells during cultivation. The experimental results also suggest that the process variables, i.e., OUR and SOUR, may be related to the accumulation of anthocyanin in Perilla frutescens cell cultures (Fig. 6). Monitoring of the above variables including OUR and SOUR during a bioreactor cultivation may be very useful for effective feeding and control in a high-density cell culture to optimize the product accumulation. The information provided by this work could be helpful for the control and optimization of plant cell bioprocesses [86]. 5.3.3 Mathematical Modeling and Process Control

Mathematical models of biological processes are often used for hypothesis testing and process optimization. Using physical interpretation of results to obtain greater insight into process behavior is only possible when structured models that consider several parts of the system separately are employed. A number of dynamic mathematical models for cell growth and metabolite pro-

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duction by plant cells have been developed for different systems by Bailey and Nicholson [100], Bramble et al. [30], Curtis et al. [101], Hooker and Lee [102], and Van Gulik et al. [103]. By taking account of culture interactions with pathways designated for structural component production, secondary metabolite synthesis and cellular respiration, a basic, structured kinetic model was successfully offered for application to batch suspension cultures of tobacco [102]. In suspension cultures of Symphytum officinale, polysaccharide production was investigated by experiments and mathematical modeling [104]. Schlatmann et al. [105] showed a simple, structured model for maintenance, biomass formation, and ajmalicine production by nondividing Catharanthus roseus cells. Several interesting parameter-control models or systems have been reported in recent years. These are a five-state mathematical model for temperature control by Bailey and Nicholson [106], a mathematical-model description of the phenomenon of light absorption of Coffea arabica suspension cell cultures in a photo-culture vessel by Kurata and Furusaki [107], and a bioreactor control system for controlling dissolved concentrations of both O2 and CO2 simultaneously by Smith et al. [108]. Because bioprocesses are time-dependent and nonlinear in nature, the enormous complexity and uncertainty of plant cell processes require a sophisticated operational logic, which cannot easily fit into the mathematical framework of the traditional control approach. Here, a physiological-state control approach, in which the current physiological state of a cell culture is monitored [86], may be a powerful method for the control of plant cell processes, because, being based on artificial intelligence methods, in particular fuzzy sets and pattern recognition theory, no conventional mathematical model is required for the synthesis of such a control system. In reality, for the design and control of microbial processes, the introduction of such a knowledge-based methodology has proven useful when working with the fragmentary, uncertain, qualitative and blended knowledge typically available for the processes [109]. 5.4 Bioprocess Scale-Up

During the scale-up of plant cell cultures, reduced productivity has often been reported [110, 111]. This reduction could be attributed to various factors such as shear stress, oxygen supply, and gas composition. As described above, plant cells are sensitive to shear stress, and oxygen supply is also significant in affecting secondary metabolite formation in plant cell cultures [23, 62, 64]. Gas exchange between the gas and liquid phases is another important factor that may affect the scale-up of plant cell cultures. In bioreactors, forced aeration is needed to supply oxygen and to improve fluid mixing. However, it may also lead to the removal of some known (such as CO2 and ethylene) or unknown gaseous compounds. Such gaseous metabolites were proven or suggested to be important for cell growth and/or synthesis of secondary metabolites in plant cell and tissue cultures [9, 64, 112, 113]. In a shake flask there is no forced aeration and gaseous metabolites can be accumulated in its headspace. Consequently, the concentrations of the dissolved gases may be quite different between shake

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flasks and bioreactors, and, for different culture systems, the effects of gaseous compounds may also be different. Schlatmann et al. [114] studied the relationship between the dissolved gaseous metabolites and the specific ajmalicine production rate in Catharanthus roseus cultures by air recirculation in a turbine reactor, and their results indicated that a higher concentration of dissolved gaseous metabolites reduced the ajmalicine production. However, in suspension cultures of Thalictrum rugosum in an airlift reactor, carbon dioxide and ethylene were shown to have stimulatory effects on alkaloid production [113]. Mirjalili and Linden [115] investigated the effects of different concentrations and combinations of oxygen, carbon dioxide and ethylene on the cell growth and paclitaxel production in suspension cultures of Taxus cuspidata on a flask scale. They found that the most effective gas mixture composition in terms of paclitaxel production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. In suspension cultures of Taxus chinensis, it was noted that a considerable drop in taxuyunnanine C (TC, a novel bioactive compound) accumulation occurred when the cells were transferred from a shake flask to a bioreactor [116]. A systematic approach was taken by performing a series of experiments in combination with theoretical analyses to identify the potential factors that may be responsible for the process scale-up [117]. Finally, ethylene incorporation into the inlet air was carried out to determine whether the reduction of taxane production in bioreactors was due to the stripping-off of ethylene during cultivation. As shown in Table 7, it is clear that the ethylene had a promoting effect on the cell growth, and the maximum biomass reached 16.3 and 18.5 g l–1 at an ethylene concentration of 6 and 18 ppm, respectively. The values were much higher than that of the control bubble column reactor, and even a little higher than the shake flasks. As to the accumulation of TC, its content was similar to the control bioreactor when 6 ppm of ethylene was incorporated into the inlet airflow, and it was obviously much less than that in the shake flasks. However, when 18 ppm of ethylene was directed into the bioreactor, the TC content was greatly enhanced and reached the same level as in the shake flasks at the end of cultivation (day 17). Therefore, although the effects of ethylene on secondary metabolism of plant cell cultures are dependent on the cell species and specific cell lines as used [113, 115, 118–120], the above example indicates that ethylene

Table 7. Cell growth and TC accumulation by Taxus chinensis cells in Erlenmeyer flasks and bubble column reactors incorporated with different levels of ethylene

Flask

Bubble column

Ethylene concentration (ppm)

0

0

Maximum biomass (g l–1) Average growth rate (d–1) Maximum TC content (mg g–1 DW) Maximum TC production (mg l–1) TC productivity (mg l–1 d–1)

14.6 0.17 13.3 178.8 10.5

11.7 0.14 6.2 70.7 5.1

6 16.3 0.19 7.0 111.4 6.6

18 18.5 0.22 14.2 229.0 13.5

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is a key factor in the scale-up of suspension cultures of Taxus chinensis from a shake flask to a laboratory bioreactor. The information obtained is very useful for a large-scale bioreactor cultivation of the plant cell.

6 Metabolic Engineering of Plant Cells for Metabolite Production Metabolic engineering of cells is a natural outcome of recombinant DNA technology. In a broader sense, metabolic engineering can be viewed as the design of biochemical reaction networks to accomplish a certain objective. Typically, the objective is either to increase the rate of a desired product or to reduce the rate of undesired side- products, or to decompose the toxic or undesired substances. Of central importance to this field is the notion of cellular metabolism as a network. In other words, an enhanced perspective of metabolism and cellular function can be obtained by considering the participating reactions in their entirety, rather than on an individual basis [121]. This research field is, therefore, multidisciplinary, drawing on information and techniques from biochemistry, genetics, molecular biology, cell physiology, chemistry, chemical engineering, systems science, and computer science [122]. To improve the productivity of commercially important compounds in plants or plant cell cultures, or even to produce completely new compounds, metabolic engineering of plant secondary metabolite pathways has opened up a new promising perspective [123, 124]. In Taxus chinensis cell cultures, Srinivasan et al. [125] examined the dynamics of taxane production and metabolic pathway compartmentation by using metabolic inhibitors, elicitors, and precursors. Potential yield-limiting steps were predicted, which was useful in facilitating the metabolic engineering of Taxus cell cultures for paclitaxel production. Verpoorte et al. [126] introduced both the genes encoding tryptophan decarboxylase and strictosidine synthase from Catharanthus roseus into tobacco, and the cells were capable of synthesizing strictosidine after feeding of secologanin. For metabolic engineering of cultured tobacco cells, Shinmyo and co-workers [127] constructed a gene expression system by isolating three cDNA clones, which showed a high mRNA level in the stationary growth phase. They claimed that promoters of these genes would be useful in gene expression in high cell-density culture. In another aspect, as suggested in the case of Chinese hamster ovary cells by Fussenegger et al. [128], coordinated manipulation of multiple genes may also be required for the effective reprogramming of complex metabolism regulatory apparatus of plant cell cycle to achieve bioprocess goals, such as cessation of proliferation at high cell density to allow an extended period of high production. In recent years, there have been many developments in biochemical and molecular-genetic studies in plants, such as biosynthetic pathways and metabolism of terpenoids [129] and cloning and expression of triterpene synthase cDNAs from triterpene-producing plants or plant cells [130]. Rapid progress in metabolic engineering of plant cells for highly efficient production of useful metabolites in cell cultures is expected in the near future. Also, by adopting metabolic engineering tools, the exploitation of the molecular diversity and con-

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trol of the heterogeneity of plant cell metabolites such as ginsenosides may be possible, which will be very meaningful to the production and application of various medicinal plants including many famous traditional Chinese herbs [131].

7 Conclusions and Future Perspectives Plant cell culture can produce many valuable metabolites including novel medicinal agents and recombinant products [132–136]. In combination with synthetic chemistry, the methodology also affords an attractive route to the synthesis of complex natural products and related compounds of industrial importance [137]. In recent years, the market for natural plant products has expanded rapidly, and this trend will continue because more and more people prefer to use natural products. Plant cell culture will contribute more to the market with the technological advancement in the future. For example, the current world market of raw materials of ginseng is about one billion US$ [138]; although cell-cultured ginseng occupies less than 1% of the market, its share will increase greatly with enhancement of the culture productivity. In this chapter, it has been demonstrated how the significance of the design and optimization of bioreactor hardware, the manipulation of culture environments including medium (e.g., sucrose, N, K+, feeding), light, shear, O2 and rheology, as well as the development of new bioreactor cultivation processes such as high-density cultivation, continuous culture, process monitoring, modeling and control, and bioprocess scale-up, can enhance the productivity of plant cell suspension cultures for useful metabolite production. There is reason to believe that a significant advance in the process control and optimization of plant cell cultures will be achieved in the near future, although at present there are two main obstacles – the lack of an adequate process monitoring and control system for plant cells and the heterogeneity and instability of the cells. Furthermore, a breakthrough in the metabolic engineering of plant cells, such as considerable improvements in cell physiology and secondary metabolism, will ultimately enhance the productivity of many plant cell processes and push them to an economically feasible stage for commercial production of useful metabolites. In the future, an intimate cooperation between biologists and biochemical engineers is necessary that will require researchers in both these disciplines to expand their knowledge bases and research fields to the hybrid cutting-edge. Acknowledgements. Financial support from the National Natural Science Foundation of China

(Project No. 29606003), the Ministry of Education of China (MOE), and the Science and Technology Commission of Shanghai Municipality is gratefully acknowledged. The author also thanks the Cheung Kong Scholars Program (CKSP) of MOE and the East China University of Science and Technology (ECUST). The experimental results from the author’s laboratory were mainly obtained by Wei-Wei Hu, Si-Jing Wang, Yi-Heng Zhang, Song Liu, Hong-Qiang Wang, Zhi-Wei Pan, and Zhi-You Wen.

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49. Kurata H, Seki M, Furusaki S, Furuya T (1991) J Chem Eng Jpn 24:783 50. Doran PM (1999) Biotechnol Prog 15:319 51. Singh G, Curtis WR (1994) In: Shargool PD, Ngo TT (eds) Biotechnological applications of plant cultures. CRC Press, Boca Raton, FL, p 151 52. Dunlop EH, Namdev PK, Rosenberg MZ (1994) Chem Eng Sci 49:2263 53. Ho CH, Henderson KA, Rorrer GL (1995) Biotechnol Prog 11:140 54. Hooker BS, Lee JM, An G (1989) Enzyme Microb Technol 11:484 55. Kieran PM, O’Donnell HJ, Malone DM, MacLoughlin PF (1995) Biotechnol Bioeng 45:415 56. MacLoughlin PF, Malone DM, Murtagh JT, Kieran PM (1998) Biotechnol Bioeng 58:595 57. Meijer JJ, ten Hoopen HJG, van Gameren YM, Luyben KChAM, Libbenga KR (1994) Enzyme Microb Technol 16:467 58. Takeda T, Kitagawa T, Takeuchi Y, Seki M, Furusaki S (1998) Can J Chem Eng 76:267 59. Kieran PM, MacLoughlin PF, Malone DM (1997) J Biotechnol 59:39 60. Steward N, Martin R, Engasser JM, Goergen JL (1999) Biotechnol Bioeng 66:114 61. Zhong JJ, Fujiyama K, Seki T, Yoshida T (1994) Biotechnol Bioeng 44:649 62. Leckie F, Scragg AH, Cliffe KC (1991) Biotechnol Bioeng 37:364 63. Kobayashi Y, Fukui H, Tabata M (1991) Plant Cell Rep 9:496 64. Gao J, Lee JM (1992) Biotechnol Prog 8:285 65. Schlatmann JE, Moreno PRH, Vinke JL, ten Hoopen HJG, Verpoorte R, Heijnen JJ (1994) Biotechnol Bioeng 44:461 66. Schlatmann JE, Vinke JL, ten Hoopen HJG, Heijnen JJ (1995) Biotechnol Bioeng 45:435 67. Zhong JJ, Seki T, Kinoshita S, Yoshida T (1992) Biotechnol Bioeng 40:1256 68. Su WW (1995) Appl Biochem Biotech 50:189 69. Rodriguez-Monroy M, Galindo E (1999) Enzyme Microb Technol 24:687 70. Pan ZW, Zhong JJ, Wu JY, Takagi M, Yoshida T (1999) Biotechnol Bioproc Eng 4:269 71. Ballica R, Ryu DDY, Powell RL, Owen D (1992) Biotechnol Prog 8:413 72. Curtis WR, Emery AH (1993) Biotechnol Bioeng 42:520 73. Zhong JJ, Yoshida T (1994) In: Ryu DDY, Furusaki S (eds) Advances in plant biotechnology. Elsevier Science, Amsterdam, p 255 74. Zhang YH, Wang HQ, Liu S, Yu JT, Zhong JJ (1997) Biotechnol Lett 19:943 75. Zhang YH (1996) MS thesis, East China University of Science and Technology 76. Zhong JJ, Yoshida T (1995) Enzyme Microb Technol 17:1073 77. Matsushita T, Koga N, Ogawa K, Fujino K, Funatsu K (1994) In: Ryu DDY, Furusaki S (eds) Advances in plant biotechnology. Elsevier Science, Amsterdam, p 339 78. Scragg AH, Ashton S,York A, Bond P, Stepan-Sarkissian G, Grey D (1990) Enzyme Microb Technol 12:292 79. Seki M, Takeda M, Furusaki S (1995) J Chem Eng Jpn 28:488 80. Seki M, Ohzora C, Takeda M, Furusaki S (1997) Biotechnol Bioeng 53:214 81. Phisalaphong M, Linden JC (1999) Biotechnol Prog 15:1072 82. Van Gulik WM, ten Hoopen HJG, Heijnen JJ (1992) Biotechnol Bioeng 40:863 83. Westgate PJ, Curtis WR, Emery AH, Hasegawa PM, Heinstein PF (1991) Biotechnol Bioeng 38:241 84. Kessler M, ten Hoopen HJG, Heijnen JJ, Furusaki S (1997) Biotechnol Tech 11:507 85. Asali EC, Mutharasan R, Humphrey AE (1992) J Biotechnol 23:83 86. Zhong JJ, Konstantinov KB, Yoshida T (1994) J Ferment Bioeng 77:445 87. Scheper TH, Lammers F (1994) Curr Opin Biotechnol 5:187 88. Taya M, Tone S, Prenosil JE (1989) Plant Tiss Cult Lett (Jap) 6:179 89. Ryu DDY, Lee SO, Romani RJ (1990) Biotechnol Bioeng 35:305 90. Tanaka H, Uemura M, Kaneko Y, Aoyagi H (1993) J Ferment Bioeng 76:501 91. Markx GH, Davey CL, Kell DB, Morris P (1991) J Biotechnol 20:279 92. Matanguihan RM, Konstantinov KB, Yoshida T (1994) Bioprocess Eng 11:213 93. Tanaka H, Aoyagi H, Jitsufuchi T (1992) J Ferment Bioeng 73:130 94. Zhong JJ, Fujiyama K, Seki T, Yoshida T (1993) Biotechnol Bioeng 42:542 95. Albiol J, Campmajo C, Casas C, Poch M (1995) Biotechnol Prog 11:88

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Jian-Jiang Zhong Smith MAL, Reid JF, Hansen AC, Li Z, Madhavi DL (1995) J Biotechnol 40:1 Miyanaga K, Seki M, Furusaki S (2000) Biotechnol Bioeng 67:493 Yanpaisan W, King NJC, Doran PM (1998) Biotechnol Bioeng 58:515 Wen ZY, Zhong JJ (1995) Biotechnol Tech 9:521 Bailey CM, Nicholson H (1989) Biotechnol Bioeng 34:1331 Curtis WR, Hasegawa PM, Emery AH (1991) Biotechnol Bioeng 38:371 Hooker BS, Lee JM (1992) Biotechnol Bioeng 39:765 Van Gulik WM, ten Hoopen HJG, Heijnen JJ (1993) Biotechnol Bioeng 41:771 Glicklis R, Mills D, Sitton D, Stortelder W, Merchuk JC (1998) Biotechnol Bioeng 57:732 Schlatmann JE, ten Hoopen HJG, Heijnen JJ (1999) Biotechnol Bioeng 66:147 Bailey CM, Nicholson H (1990) Biotechnol Bioeng 35:252 Kurata H, Furusaki S (1993) Biotechnol Prog 9:86 Smith JM, Davison SW, Payne GF (1990) Biotechnol Bioeng 35:1088 Shioya S, Shimizu K, Yoshida T (1999) J Biosci Bioeng 87:261 Schlatmann JE, Nuutila AM, Van Gulik WM, ten Hoopen HJG, Verpoorte R, Heijnen JJ (1993) Biotechnol Bioeng 41:253 Scragg AH, Morris P, Allan EJ, Bond P, Fowler MW (1987) Enzyme Microb Technol 9:619 Biddington NL (1992) Plant Growth Regul 11:173 Kim DI, Pedersen H, Chin CK (1991) Biotechnol Bioeng 38:331 Schlatmann JE, Moreno PRH, Vinke JL, ten Hoopen HJG, Verpoorte R, Heijnen JJ (1997) Enzyme Microb Technol 20:107 Mirjalili N, Linden JC (1995) Biotechnol Bioeng 48:123 Wang HQ (1998) PhD thesis, East China University of Science and Technology Pan ZW, Wang HQ, Zhong JJ (2000) Enzyme Microb Technol 27:714 Cho GH, Kim DI, Pedersen H, Chin CK (1988) Biotechnol Prog 4:184 Lee CWT, Shuler ML (1991) Biotechnol Tech 5:173 Songstad DD, Giles KL, Park J, Novakowski D, Epp D, Friesen L, Roewer I (1989) Plant Cell Rep 8:463 Stephanopoulos G (1994) Curr Opin Biotechnol 5:196 Shimizu K (2000) In: Zhong JJ (ed) Advances in applied biotechnology. ECUST Press, Shanghai, p 3 Verpoorte R, Van Der Heijden R, ten Hoopen HJG, Memelink J (1999) Biotechnol Lett 21:467 Shanks JV, Bhadra R, Morgan J, Rijhwani S, Vani S (1998) Biotechnol Bioeng 58:333 Srinivasan V, Ciddi V, Bringi V, Shuler ML (1996) Biotechnol Prog 12:457 Verpoorte R, Van der Heijden R, Memelink J (1996) Phytother Res 10:S12 Shinmyo A, Shoji T, Bando E, Nagaya S, Nakai Y, Kato K, Sekine M, Yoshida K (1998) Biotechnol Bioeng 58:329 Fussenegger M, Schlatter S, Daetwyler D, Mazur X, Bailey JE (1998) Nat Biotechnol 16:468 McCaskill D, Croteau R (1998) TIBTECH 16:349 Ebizuka Y, Kushiro T, Shibuya M (1999) Intl Ginseng Conf ’99, Hong Kong Zhong JJ (1999) Proceedings of the 5th Asia-Pacific Biochem Eng Conf. Phuket, Thailand Kim SI, Choi HK, Kim JH, Lee HS, Hong SS (2001) Enzyme Microb Tech 28:202 Wang CG, Wu JY, Mei XG (2001) Biotechnol Prog 17:89 Stoner MR, Humphrey CA, Coutts DJ, Shih NJR, McDonald KA, Jackman AP (1997) Biotechnol Prog 13:799 Fowler MW (1992) In: Coombes JD (ed) New Drugs Mat Sources. IBC Tech Serv, London, p 131 James E, Lee JM (2001) this volume Kutney JP (1996) Pure Appl Chem 68:2073 Wu J, Zhong JJ (1999) J Biotechnol 68:89

Received September 2000

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Gas Concentration Effects on Secondary Metabolite Production by Plant Cell Cultures J. C. Linden, J. R. Haigh, N. Mirjalili, M. Phisaphalong Department of Chemical and Bioresource Engineering, Colorado State University, Fort Collins, CO 80523, USA, e-mail: [email protected]

One aspect of secondary metabolite production that has been studied relatively infrequently is the effect of gaseous compounds on plant cell behavior. The most influential gases are believed to be oxygen, carbon dioxide and other volatile hormones such as ethylene and methyl jasmonate. Organic compounds of interest include the promising antimalarial artemisinin (known as “qing hao su” in China where it has been a folk remedy for centuries) that is produced by Artemisia annua (sweet wormwood) and taxanes used for anticancer therapy that are produced by species of Taxus (yew). The suspension cultures of both species were grown under a variety of dissolved gas conditions in stoppered culture flasks and under conditions of continuous headspace flushing with known gas mixtures. An analysis is presented to show the culture conditions are such that equilibrium between the culture liquid and gas headspace is assured. The growth rate of the cells and their production rates of artemisinin and paclitaxel were determined. These and other parameters are correlated as functions of the gas concentrations. Interdependence of ethylene and methyl jasmonate is also explored with respect to regulation of secondary metabolite formation. Keywords. Plant cell culture, Secondary metabolites, Oxygen, Carbon dioxide, Ethylene, Methyl

jasmonate, Artemisinin, Paclitaxel, Engineering parameters

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

1.1 1.2 1.3 1.3.1 1.3.2 1.3.3 1.4 1.4.1 1.4.2

Examples of Secondary Metabolites Produced by Plant Cell Culture Comparison with Well-Established Microbial Fermentations . . . . Importance of Dissolved Gases as Medium Components . . . . . . . Specific Consumption of Oxygen . . . . . . . . . . . . . . . . . . . . Specific Productivity of Carbon Dioxide . . . . . . . . . . . . . . . . Specific Productivity of Ethylene . . . . . . . . . . . . . . . . . . . . Review of Relevant Engineering Parameters . . . . . . . . . . . . . . Mass Transfer Considerations in Entrapped Culture Systems . . . . Dissolved Gases in Suspension Culture Systems . . . . . . . . . . .

2

Artemisia annua Production of Artemisinin: Dependence on Dissolved Gas Concentrations . . . . . . . . . . . . . . . . . . . . 37

2.1 2.2 2.3 2.4

Initial Specific Gas Usage and Productivities . . . . . . . . . . . . . Absence of Significant Mass Transfer Resistance . . . . . . . . . . . Calculation of Specific Oxygen Usage Rate Throughout a Test . . . Effect of Headspace Composition on Growth and Production of Artemisinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

28 29 30 31 31 31 32 32 35

38 41 42 43

Advances in Biochemical Engineering/ Biotechnology, Vol. 72 Managing Editor: Th. Scheper © Springer-Verlag Berlin Heidelberg 2001

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3

Taxus cuspidata and Taxus canadensis Production of Paclitaxel: Dependence on Dissolved Gas Concentrations . . . . . . . . . . . . 48

3.1

Optimization of Headspace Composition on Production of Paclitaxel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Application of Volatile Methyl Jasmonate to Headspace Flow . . . . 50 Formation of Ethylene and Methyl Jasmonate Upon Elicitation of Plant Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . 51

3.2 3.3 4

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

1 Introduction Plants provide a wide variety of biochemicals useful to humanity. Their uses include medicinal compounds, flavors, fragrances and agricultural chemicals. A number of investigators have studied the use of plant cells in culture, rather than whole plants, as sources of some of the more valuable organic compounds. Before such processes can become a viable manufacturing option, a great deal more must be learned about the optimum conditions for growth and productivity of cells in culture. The use of large quantities of individual cells in a controlled environment is the basis of the well-established fermentation industry. In most fermentation processes, microbial cells (bacteria or fungi) of a particular species are grown in large-scale suspension cultures. Conditions such as temperature, pH and concentrations of dissolved oxygen, carbon source and other nutrients are controlled in order to maximize the production rate of the desired compound. Productivities of commercial fermentation products have been enhanced orders of magnitude above those exhibited in nature. The same principles need to be applied to plant cell cultures. 1.1 Examples of Secondary Metabolites Produced by Plant Cell Culture

The production of commercial products from plant cell culture processes has been very slow to occur. Although the matter has been discussed in the scientific literature for well over three decades [1], only a few commercial-scale processes have even been attempted that use plant cell culture to produce secondary metabolites [2]. In Japan three submerged fermentation processes using plant cell cultures were developed by Mitsui Petrochemical Industries to produce berberine, ginseng and shikonin on scales from 4000 to 20,000 l. Only one of these, production of shikonin by suspension cultures Lithospernum erythrorhizon, can be considered a clear success. It has been operated since the early 1980s [3]. Difficulty in obtaining regulatory approval for use of these products as medicinals has impeded commercialization. In North America commercial production of sanguinarine and vanilla flavor was attempted but failed for re-

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Gas Concentration Effects on Secondary Metabolite Production by Plant Cell Cultures

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gulatory reasons. However, Phyton (Ithaca, NY) utilizes a 75,000-l reactor at a facility near Hamburg, Germany, to develop commercial production of paclitaxel [4]. In addition to food and fiber, plants are exploited for a large variety of commercial chemicals, including agricultural chemicals, pharmaceuticals, food colors, flavors and fragrances [5]. A few examples of plant-derived pharmaceuticals are digitalis (produced from Digitalis purpurea, prescribed for heart disorders), codeine (Papaver somniferum, sedative), vinblastine and vincristine (Catharanthus roseus, leukemia treatment) and quinine (Cinchona officinalis, malaria) [1]. It is believed that plant tissue culture production methods (called “phytoproduction”) can be developed to profitably manufacture some of these chemicals [1–9]. Encouraged by these advantages, Routian and Nickell obtained the first patent for the production of substances by plant tissue culture in 1956 [10]. More recent patents cover many aspects of using plant cell culture for secondary metabolite production [11–13]. Numerous investigators have reported production of useful compounds in hairy root, callus and suspension cultures. The diversity of plant materials adaptable to culturing in hairy root and callus cultures has recently been reviewed [14, 15]. Suspension cultures of Thalictrum minus produced the stomachic and antibacterial berberine [16]. Callus cultures of Stizolobium hassjo produced the antiparkinsonian drug Ldopa [17]. Suspension cultures of Hyoscyamus niger L. produced a derivative of the anticholinergic hyoscyamine [18]. Some secondary metabolites have been observed in much higher concentrations in cultured cells than in whole plants of the same species. These include ginsengosides from Panax ginseng (27% of cell dry weight in culture, 4.5% in whole plants), anthraquinones from Morinda citrafolia (18% in culture, 2.2% in plants) and shikonin from Lithospernum erythrorhizon (12% in culture, 1.5% in plants) [19, 20]. Additional examples of substances synthesized by cell culture are listed in review articles and books [1–8, 14, 15, 21]. It is important to note that many of these compounds are secondary metabolites; that is, their production is not related to cell growth and division. Indeed, high rates of production of secondary metabolites usually occur during low rates of growth, often under conditions of significant physiological or biochemical stress on the cells [4]. 1.2 Comparison with Well-Established Microbial Fermentations

Phytoproduction holds several difficulties compared with the well-established microbial fermentations [1, 4, 6, 22, 23]: 1 Plant cells grow much more slowly, with doubling times of the order of 40 h (compared with 0.3 h for some bacteria). Consequently, costs associated with cell generation are much greater. 2 Specific production rate tends to be lower. For example, despite several years of optimization studies, volumetric productivity of shikonin by suspension cultures of L. erythrorhizon was reported as 0.1 gproduct l–1 d–1 [23]. For com-

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parison, the fungus Penicillium crysogenum yields 3.2 g l–1 d–1 of penicillin [24]. 3 Plant cells may store their products in vacuoles rather than secrete them into the medium. 4 Plant cells are much more sensitive to shear forces than are bacteria or yeast cells, requiring much gentler aeration and agitation. The medium in contact with cultured plant cells must consist of a large number of components for growth to occur. For the purpose of the present discussion, these components may be categorized as follows: 1 2 3 4 5 6 7 8

Water Carbon source Concentrated inorganic salts, including nitrogen sources Trace salts (usually considered to be those of less than 0.1 g l–1 in the medium) Vitamins Plant hormones and cytokinins Medium conditioning factors (compounds produced in very small quantities by the cells themselves) Dissolved gases

Clearly, the first six component types can be controlled during the initial formulation of the medium. It is these that have been the subject of optimization studies, which form a large part of the recent plant cell culture literature. The seventh category falls outside the capabilities of most investigators in the field. The concentrations of dissolved gases have also been neglected as components, possibly because they cannot be controlled in the same manner as dissolved solids. 1.3 Importance of Dissolved Gases as Medium Components

In the present study, we attempted to determine the effects of important dissolved gases, oxygen, carbon dioxide and ethylene, on growth of species of three plant genera, Nicotiana, Artemisia and Taxus, in culture. The productivity of respectively valuable metabolic compounds, generic phenolic compounds, artemisinin and paclitaxel, and aspects of culture physiology were also studied. Artemisinin is a promising antimalarial drug and paclitaxel is an effective anticancer drug, so progress towards improved methods of manufacturing would be very desirable. Moreover, N. tabacum, A. annua, T. cuspidata and T. canadensis can be considered as model systems; the methods (and some of the conclusions) developed from the current studies may be applicable to other phytoproduction systems [25–30]. In an efficient commercial phytoproduction process, the cells will be in contact with near optimum concentrations of each dissolved gas. However, to insure that this occurs, the designer must know both the ideal concentrations and (for material balance reasons) the production and usage rates.

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1.3.1 Specific Consumption of Oxygen

Both the studies of Hulst et al. [31] and Hallsby [32, 33] reported zero-order consumption; that is, in the normal concentration range, cellular O2 consumption was not dependent on its concentration. LaRue and Gamborg measured O2 –1 h–1 over the life of a culture of rose cells, but did not inusage of 0.03 mmol gdw vestigate the order of reaction [34]. Taticek et al. tabulated several values of maximum specific O2 usage rates, –1 [6]. Our experiments with A. annua which range from 0.2–0.6 mmol g–1 dw h suspension cell cultures indicate a maximum specific usage rate of –1 0.2 mmol g–1 dw h . The wide variation in usage rates is suspicious; it may be an artifact of the variety of methods of estimation. For comparison, several specific usage rates for industrially important bacteria and fungi are given. These range from 3.0 (Aspergillis niger) to –1 10.8 mmol g–1 dw h (Escherichia coli) [24]. 1.3.2 Specific Productivity of Carbon Dioxide

Thomas and Murashige investigated several solid callus cultures of several plant species, but did not test any suspension cultures [35]. Concentrations of CO2, ethylene and some other gases were measured 24 h after flushing with air. Zobel measured CO2 and C2H4 evolution from soybean callus cultures [36]. Fujiwara et al. measured CO2 concentrations in closed vessels containing cultured plantlets [37]. None of these reports give enough information to permit the calculation of specific productivity. 1.3.3 Specific Productivity of Ethylene

Ethylene (C2H4) is produced in essentially every part of every seed plant and affects a number of metabolic functions in very small concentrations. It is therefore considered a plant hormone [38]. Cultured plant cells are also known to produce C2H4 . The highest known ethylene release rate is by fading flowers of Vanda –1 [39]. In some of the orchids, producing approximately 3 ¥ 10–3 mmol g–1 dw h sources mentioned in the previous section, both CO2 and C2H4 concentrations were measured, but, again, not enough data is reported to obtain productivity values. LaRue and Gamborg report the amounts of C2H4 produced by suspension cultures of several species [34]. The time course data they present shows that C2H4 production is very unsteady; however, average productivities can be estimated. For soybean and rose cultures, for example, productivities were 1 ¥ –1 10–6 and 1.4 ¥ 10–5 mmol g–1 dw h , respectively. Most of the other species tested lay between those results. Lieberman et al. measured ethylene productivities of callus and suspension cultures of apple [40]. For callus cultures, productivity –1 peaked at 2.1 ¥ 10–6 mmol g–1 dw h , and averaged about two-thirds that value. For

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–1 suspension cultures, productivity peaked at 9.4 ¥ 10–6 mmol g –1 dw h , but aver–6 –1 –1 aged 2.7 ¥ 10 mmol g dw h .

1.4 Review of Relevant Engineering Parameters

In a review article advocating entrapped plant cell cultures, Shuler et al. [41] point out the problem in large-scale suspension cultures of maintaining sufficient oxygen transfer without excessive mechanical shear on the cells. A process designer must not overdesign for oxygen transfer because this would result in increased cell damage. Conventional bioreactors are completely back-mixed stirred tank reactors (STR) that deliver oxygen and remove carbon dioxide by sparging air through the medium. Conventional STR bioreactors exhibit oxygen transfer rates (OTRs) of between 5–150 mmol O2 h–1 l–1 under normal operating conditions. When the growth-limiting nutrient is oxygen in batch culture, cells grow exponentially until the oxygen uptake (or consumption) rate (OUR) exceeds the oxygen delivery rate (OTR) of the bioreactor. The point at which oxygen becomes the limiting nutrient and the OTR dictates the growth rate can be illustrated at various OTRs using the exponential growth and yield expressions for growth: xt = x0 emt

(1)

Dx = Yx/s DS

(2)

where m is the specific growth rate (h–1) (which is constant until such time that nutrient becomes limiting, a product becomes toxic, or culture conditions change), t is time (h), x is cell dry weight (g l–1) at the initial condition, 0, and after time t, Yx/s is the yield coefficient, and S is substrate concentration (g l–1). 1.4.1 Mass Transfer Considerations in Entrapped Culture Systems

Oxygen limitation is of concern in entrapped as well as suspension cultures; for example, many investigators have found it necessary to guarantee sufficient aeration by installation of oxygen-permeable silicone tubing (with oxygen pressure on the inside of the tube) in immobilized cell bioreactors. The O2 flux rate is calculated and judged more than sufficient for the entrapped cells, but no estimate of the actual requirement was presented. In multi-phase bioreactors resistance to oxygen transfer is the sum of resistances in the gas-phase viscous boundary layer, the liquid-phase viscous boundary layer and the immobilized cell surfaces. Generally, however, in cases of several resistances in series, only one of the resistances is large enough (relative to the others) to control the transport rate and is referred to as the rate-limiting resistance. A Wilson plot (reciprocal of mass transfer coefficient vs. the reciprocal of linear fluid velocity) can be used to determine which is the rate-limiting resistance to oxygen transfer. Extrapolation of such double reciprocal plots to the y-axis yield mass transfer rates at infinite velocity, where there would be no boundary layer resistance.

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Significant effort on immobilized plant cell [9, 25, 42] and transformed hairy root [43–45] cultures has been expended by several groups of chemical engineering researchers. The growth of Agrobacterium-transformed “hairy root” cultures of Hyoscyamus muticus, examined in various liquid- and gas-dispersed bioreactor configurations, has been studied by McKelvey et al. [46]. Accumulated tissue mass in submerged air-sparged reactors was 31% of that in gyratory shake-flask controls. Experiments demonstrated that poor performance of sparged reactors was not due to bubble shear damage, carbon dioxide stripping, settling or flotation of roots. Impaired oxygen transfer due to channeling and stagnation of the liquid phase was the apparent cause of poor growth. Hulst and co-workers obtained a value for the O2 uptake in both suspended and calcium alginate immobilized Daucus carota (carrot) cells [31]. This value –1 was approximately 3 ¥ 10–7 mol g–1 dw s . Nevertheless, the value of Hallsby’s esti–10 –1 –1 mate of uptake of oxygen, 3 ¥ 10 g–1 dw mol s for N. tabacum, will be used [32, 33]. Hulst’s observation that specific consumption was similar for suspended and immobilized cells is assumed to apply [31]. During most of our studies with calcium alginate immobilized N. tabacum cells, the bioreactor columns contained about 0.04 g (dry weight) of cells; of course, this value varied from test to test. It is reasonable to assume that the feed medium was saturated in O2 . The O2 mole fraction, x, in the medium (at saturation) is given by Henry’s Law [47]: x = p/H

(3)

where p is the partial pressure of O2 in the aeration air (in atmospheres) and H is Henry’s Law constant, reported at 25 °C to be 4.4 ¥ 104 (in units consistent with x and p). The latter value is nearly independent of pressure. The atmospheric pressure of our laboratory (and hence the O2 partial pressure in the aeration air) is affected by its elevation, approximately 1500 m above sea level. The average total pressure is 846 millibars, or 0.835 atmosphere [48]. Air is 21% O2 , so the O2 partial pressure in the saturated medium, x = 0.835 atm ¥ 0.21/(4.4 ¥ 104 atm mole fraction–1) = 4.0 ¥ 10–6 [mole fraction O2]. Converting from mole fraction to concentration units by multiplying by the molar concentration of liquid water, the O2 concentration is: 4 ¥ 10–6 ¥ 0.056 mol cm–3 = 2.2 ¥ 10–7 mol cm–3 The flow rate for most tests was of the order of 0.5 cm3 h–1. This resulted in a total feed rate of O2 of 3.1 ¥ 10–11 mol s–1, less by a factor of 10 than the estimated requirement. Suppose it is correct that O2 consumption is a zero-order process until its concentration is depleted to 15% of saturation (at sea level). Then 80–85% of the available oxygen was consumed by approximately 10% of the cells – the 10% with which the feed first came in contact. Clearly, the remainder of the cells was starved for oxygen. The resulting biochemical stress could have effected an increase in productivity of phenolics; indeed, Hallsby concluded from batch ex-

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periments that a suboptimum level of O2 may be advantageous for the formation of extracellular phenolics [31]. However, it is likely that most cells were severely restricted in metabolism (both primary and secondary) or died due to lack of oxygen. The decline in productivity that we observed suggests death of most of the cells, although it is surprising (and not conclusively explained) that this decline occurred over such a long period. One would have expected that the productivity would drop rapidly to 10% of the initial value and remain there. The following discussion centers on the particle effectiveness factor of calcium alginate immobilized cells. Other investigators have estimated the effective diffusivity of oxygen in this material. This parameter can have, as will be shown, a significant effect upon whether any of the cells are starved for oxygen. If sufficient O2 had been supplied to the alginate beads, there would not have been diffusion limitation in the beads, as evidenced by the following. First, it will be shown that the oxygen concentration in the medium at the bead surface very nearly equals the bulk concentration. Then it will be shown that this concentration is sufficient to achieve an effectiveness factor of unity for the zeroorder process believed to occur. The steady-state diffusion rate from a stagnant medium of infinite extent to a sphere is given by: Flux = (2D/d) (cB – co)

(4)

where D is the diffusivity in the bulk fluid, d is the bead diameter, and co and cB are the concentrations of the diffusing species at the bead surface and in the bulk liquid, respectively [49]. Diffusive flux is the rate of flow of the species through the surface per unit area. Thus, at steady state, the product of the flux and the surface area equals the consumption rate within the bead. This equality is: (5) (pd2)(2D/d)(cB – c0) = (d3p/6) bk where the right side represents the bead volume times the biomass density times the consumption rate per unit biomass. Rearranging, cB – co = (d2 bk)/12D

(6)

The approximate parameter values are: d = 0.5 cm, b = 2 ¥ 10–4 gdw cm–3, k = 8 –1 s–1, and D = 2.4 ¥ 10–5 cm2 s–1, giving: ¥ 10–9 mol g dw cB – co = 1.4 ¥ 10–9 mol cm–3. This compares to a cB of 2 ¥ 10–7 mol cm–3, so the surface concentration is within 1% of the bulk liquid concentration. For a zero-order process, the effectiveness factor (defined as the ratio of the actual consumption of reactant to that which would occur in the absence of mass transfer resistance) is equal to unity whenever the following inequality holds: (7) d < (24 Deff co/kb)1/2 Here d is the bead diameter (cm), k is the intrinsic rate constant (moles consumed/s per gram dry weight of biomass), b is the biomass per bead volume

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(gdw cm–3), Deff is the effective diffusivity (cm2 s–1) of oxygen in a bead, and co is the O2 concentration (mol cm–3) at the bead exterior. Hulst estimates the effective diffusivity of O2 in a calcium alginate bead to be 1.9 ¥ 10–5 cm2 s–1 [31]. Near the inlet of the column, c0 = 2 ¥ 10–7 mol cm–3 (see above). The rate con–1 s–1 (this is Hallsby’s consumption estistant k is estimated as 8 ¥ 10–9 mol g dw mate as used above). The biomass per bead volume was typically 2 ¥ 10–4 gdw cm–3. The critical diameter for these conditions is 7.5 cm. Even if there are large uncertainties in the rate parameters, the critical diameter is much larger than the actual diameter of 0.5 cm. 1.4.2 Dissolved Gases in Suspension Culture Systems

As mentioned in the previous section, at least two investigators observed zeroorder dependence of O2 consumption on its concentration. Hallsby claims this is true for suspended tobacco cells whenever O2 concentration exceeds 15% of the concentration in water in equilibrium with air (the “air saturation concentration”) [33]. Hulst et al. assert that experimental values for K¢m (the O2 concentration at which O2 usage is half the usage at very high concentrations) are between 2 and 20% of air saturation [31]. At concentrations much above K¢m , consumption would be zero order. If O2 consumption were indeed zero order for a particular plant species, then it would appear that any phytoproduction process involving that species would require only that a minimum dissolved O2 concentration be maintained; any concentration increase beyond that would be irrelevant. In the case of tobacco cells, any concentration greater than 15% of air saturation would yield the same metabolic rate and, presumably, the same productivity of all metabolites. If, on the other hand, consumption is first order in the concentration range achievable in a practical bioreactor (equivalently, if K¢m is comparable to working concentrations), then its concentration is an important control parameter in the bioreactor. However, Kobayashi et al. studied berberine production by suspended and immobilized cells of Thalictrum minus [50]. They assert that O2 uptake is a zero-order process but observed that berberine production depended on O2 availability. They controlled that availability by adjusting the speed of shaking of the culture flasks, thus varying the mass transfer coefficient for absorption of O2 . Tate and Payne fed controlled concentrations of O2 and CO2 through the headspaces of suspension cultures of Catharanthus roseus, and reported a halfsaturation constant for growth (the O2 concentration at which growth was half that occurring at abundant O2) of 16% of air saturation [51]. They also observed suppressed growth at O2 concentrations above 70% in the gas phase (350% of air saturation) but did not observe that phenomenon in Daucus carota cells. Su and Humphrey grew Anchusa officinalis in a “perfusion bioreactor”, a fermentor in which an oxygen/air mix was fed through microporous tubing rather than by bubbling [52]. They plotted dissolved O2 concentration (minimum of 6% air saturation) and reported that this concentration remained “at a nonlimiting level”.

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Carbon dioxide is, of course, fundamentally important to plants because of photosynthesis. Most plant cell cultures are heterotrophic, non-photosynthetic and use a chemical energy source. It is reasonable to suspect, however, that some of the control mechanisms for the photosynthetic dark reactions would be regulated by CO2 concentration. This could affect both cell growth and, indirectly, production of useful compounds. More concretely, CO2 is known to promote synthesis of ethylene [38]; on the other hand, CO2 concentrations of 5–10% inhibit many ethylene effects [53]. A few studies on cultured plant cells have shown significant effects of CO2 . Using an airlift fermenter, Fowler observed that supplying CO2 to a tobacco cell culture increased biomass growth [54]. Maurel and Paeilleux found the same using Catharanthus roseus cells [55]. Stuhlfauth et al. found it increased secondary metabolite production in Digitalis lanata [56]. In headspace gas fed shake flasks, Tate and Payne also looked for an effect of CO2 concentration on cell growth and found none [51]. Kim et al. grew Thalictrum rugosum cells in an airlift fermenter and found that addition of CO2 to the air feed had no effect on cell growth but increased formation of berberine, the desired product [57]. A significant concern involves aerated systems, as one would expect to use in a large-scale process [58]. Preliminary studies typically involve growth in small shake flasks with limited gas exchange. When the process is scaled up to an aerated bioreactor, the aeration may strip volatile compounds, especially CO2 and C2H4, produced by the cells and which are necessary to some extent for growth and productivity. If changes in performance occur upon scale-up, it may be difficult to determine the cause. Ethylene has been shown to have a variety of effects on living plants. Thirtyfive distinct functions were tabulated by Abeles, based on literature reports existing in 1973 [59]. The majority of cases showed threshold C2H4 activity at 0.01 ppm, half-maximal effect at 0.1 ppm, and saturation (that is, further increase in C2H4 concentration did not increase the effect) at about 10 ppm. The parts per million concentrations Abeles reports are the gas-phase concentrations. In most cases, there was no further effect beyond the saturation concentration, although there were a few instances of the effect being reversed at higher concentrations [28, 60]. A number of investigators have added ethylene to plant cell cultures to enhance the yield of desired compounds. Bagratishvili and Zaprometov periodically added small quantities of 2-chloroethylphosphonic acid (commonly called ethephon, trade name Ethrel) to suspension cultures of Camellia sinersis [61]. In aqueous solution, ethephon breaks down to release ethylene, HCl and phosphate. Growth was reduced by 20% but production of phenolics and flavans increased by 90 and 75%, respectively. Songstad et al., studying sanguinarine production by suspension cultures of Papaver somniferum, observed an increase in C2H4 production occurring at the same time as sanguinarine production [62]. This led them to add Ethrel to test cultures, but it did not enhance productivity. Kobayashi et al. produced berberine in suspension cultures of Thalictrum minus and enhanced production by addition of Ethrel but reduced it greatly by addition of silver thiosulfate, a known potent inhibitor of C2H4 activity [63]. Kim et al. grew the same organism in an airlift bioreactor and improved berbe-

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rine productivity by addition of both CO2 and C2H4 to the gas feed [57]. The same group grew cultures in numerous shake flasks, allowing them to try several combinations of several added substances including Ethrel [64]. For example, their most effective combinations involved Ethrel and CuSO4 addition and a two-stage culture with high sucrose concentration in the production stage. Cho et al. enhanced production of purine alkaloids by Coffea arabica cells by addition of Ethrel and other strategies [65].

2 Artemisia annua Production of Artemisinin: Dependence on Dissolved Gas Concentrations Malaria is one of the world’s most serious human health problems. According to the World Health Organization, more than 200 million new infections occur each year, many resulting in death [66]. The disease is caused by protozoa of the genus Plasmodium, most notably P. falciparum, which live in the intestines of female Anopheles mosquitoes. Humans are infected by bites from infected mosquitoes, by blood transfusions from infected donors, or by an expectant mother transmitting the disease to her child. Malaria is endemic in Sub-Saharan Africa, Central and South America, the Indian subcontinent and Oceania [67]. For many years, the treatment of choice for malaria has been chloroquine. Unfortunately, chloroquine-resistant strains of Plasmodium species have developed, emphasizing the need for new antimalarials. One promising antimalarial is artemisinin. Beginning in 1967, the government of the People’s Republic of China conducted a systematic study of native plants used as folk remedies for a variety of ailments [67]. The herb known to the Chinese as “qing hao” (Artemisia annua, commonly called sweet wormwood or annual wormwood) was first mentioned in writings from 168 BC as a remedy for hemorrhoids. Since then, it has been recommended to relieve fevers (in 340 AD) and specifically for malaria symptoms (in 1596). In the 1970s, the activity against malaria was confirmed; the active ingredient was isolated and its structure identified. It was given the name “qing hao su” (“active ingredient in qing hao”) and in the West is now known as artemisinin. Artemisinin is a sequiterpene lactone with an endoperoxide bridge. Concentrations in plant biomass as high as 0.5% (weight of artemisinin divided

Structure 1. Artemisinin

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by weight of dried leaves or flowers) were obtained from A. annua plants growing in the Sichuan province of China. A. annua grows wild in many places, including the United States, but artemisinin contents in plants outside of China tend to be much lower. Thousands of patients in China and Myanmar (formerly Burma) have been successfully treated but, as of 1990, artemisinin is unavailable elsewhere. Relatively low toxicity has been observed, along with substantial effectiveness. Unfortunately, the relapse rate is high. Nevertheless, use of artemisinin or its derivatives in conjunction with other treatments is considered promising [67]. The small concentrations of artemisinin in plant material (and the possibility of an embargo of Chinese plant material) has led several groups of researchers to investigate use of plant cell cultures as a source of artemisinin [26, 68–70]. The results have generally been disappointing and tended to reflect some of the problems of other phytoproduction studies. Nevertheless, the results of such studies, including this one, can guide the development of phytoproduction using this and other plant species. 2.1 Initial Specific Gas Usage and Productivities

Eighteen cultures of A. annua were subjected to stoppered culture tests, which will be described below. The intention was to determine the specific productivities of CO2 and consumption rate of O2 under various conditions. To obtain the “initial” rate of formation (or usage) of any gas, its change in gas-phase concentration over the first 24 h of the test (or as near as permitted by the analysis times) was divided by the estimated biomass (dry weight basis) during that period and by the time period. The actual equation along with its development will be presented below. Ideally, we would like to obtain the instantaneous rate (of usage or production) as soon as the rubber stopper is applied. As soon as the gas composition in the headspace (hence also, the concentration of dissolved gases in the medium) changes, the behavior of the cells starts to change. However, there is, of course, uncertainty in measurement of the gas concentrations. If samples taken at a short time interval were used in the productivity calculation, the subtraction of two uncertain values close to each other would make the calculated productivity unacceptably inaccurate. The 24-h period was chosen to obtain a period of relatively large change in gas concentrations while not drastically altering the metabolic conditions during the period of the rate calculation. The quantities of gaseous CO2, O2 and C2H4 in the headspace of the stoppered culture flask were calculated from the measured concentrations. The quantity of each dissolved gas in the liquid can be estimated as described below using Henry’s Law. The total volume of the stoppered flask is 550 ml. The volume Vl of liquid and cells is 83 ml, leaving 467 ml of headspace (Vg). The atmospheric pressure (P) at Fort Collins, Colorado, where the experiments took place, is roughly 0.835 atm; any change in pressure inside the flask after the start of the experiment is negligible. Temperature (T) in our laboratory averaged

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298 K. The total amount (N, in moles) of headspace gas in each culture can be determined from the Ideal Gas Law. N = (PVg)/RT = [(0.835 atm)(.467 l) ]/[(0.082 l atm = 0.0160 moles

(8) mol–1

K–1)(298

K)]

The total number of moles, Ni , of species i in the headspace is given by Ni = 0.0160 yi

(9)

yi being the measured headspace mole fraction of i. It is necessary to estimate the quantity of each gas in the liquid to accurately determine the productivities and usage rates. The species’ equilibrium concentration, xi , in the liquid is estimated by Henry’s Law (Eq. 3). Unfortunately, H, the Henry’s Law constant, for a gas in contact with a solution depends on the nature and concentrations of dissolved solids, tending to be less than the value for pure water [71]. For this reason, we can only obtain an upper limit for the dissolved gas quantity. However, the solubility depression for our rather dilute culture medium is low. A 0.5 mole/l concentration of sodium chloride results in an oxygen solubility depression of 15% [71]. The total concentration of dissolved solids in our medium was less than half of that (0.22 mole/l), so the gas solubility depression was almost certainly less than 10%. A more serious uncertainty occurs because the culture volume includes cell volume; by treating the entire 83 ml as liquid volume (Vl), we may tend to overestimate the dissolved gas quantity. The quantity (xi Vl) is the number of moles of i dissolved in the liquid. By combining Eqs. (8) and (9), and using the known values of P and Vl , we get: (xi Vl) = Pyi Vl/H =Ni (4.33 l atm mol–1)/H

(10)

For CO2 , H is approximately 31.6 mol l–1 atm–1 [47], so (xCO2 Vl) = 0.14 NCO2

(11)

Aqueous CO2 , unlike the other gases we will consider, can further react to form other species, H2CO3 , HCO3– and CO2– 3 . The relative concentrations of the four aqueous species are a function of pH [72]. The pH of the A. annua cultures remained below 6.0. Under these conditions, the ratio of [HCO3–] to the sum of [H2CO3] and [CO2] remained less than 0.43; [CO2– 3 ] was negligible (the brackets indicate liquid phase concentration). The total value of the number of moles of dissolved CO2 and related species was always, therefore, less than 20% of Ni (14% plus 0.43 ¥ 14%). For lack of a more precise estimate, we calculate the total CO2 in both phases as 1.2 times that in the gas phase. It turns out that the other gases, O2 and C2H4 , are less soluble so their liquidphase quantities may be neglected in comparison to the gas phase. For O2 , H = 790 atm l mol–1, so xO2Vl = 0.005 ¥ NO2 . For C2H4 , H = 205 atm l mol–1, so xC2H4 Vl = 0.021 ¥ NC2H4 [47]. Clearly, the liquid-phase quantities of both of these can by neglected compared to the gas phase. The rate quantity of interest for each gas species is the specific productivity (for O2, the specific usage rate), i.e. the amount of gas produced (or used) per

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unit time per unit biomass. The amount of biomass at any given time is estimated from the initial and final biomass by assuming that growth is proportional to oxygen usage. Thus, (%O2 (0) – %O2 (t)) X (t) = X (0) + 0003 · (X (f) – X (0)) (%O2 (0) – %O2 (f))

(12)

Here, X(t), X(0) and X( f) are the quantities of biomass (dry weight) at time t, the start of the test and the finish of the test, respectively. Similarly, %O2(t), %O2(0) and %O2( f) are the measured O2 percentages in the headspace at those times. This calculation could not be performed for the tests started with cultures that had grown under normal closures before being flushed and stoppered, since the biomass at the start of the test was unknown. The X(t) values for those tests were estimated by working backwards from their final biomass, assuming that the ratio of (biomass generated)/(O2 consumed) was similar to those of the other tests. The X (t) values for the 7- and 8-day-old cultures are consequently much less reliable. The initial specific O2 usage rate, sO2 (in millimoles per hour per gram of dry biomass), is thus estimated as: (%O2 (0) – %O2 (t)) s02 = 0003 · 0.160 (t-0) · X(t/2)

(13)

where X (t/2) is the average biomass quantity over the initial period. The constant comes from the total quantity of headspace gas, 16 millimoles, corrected for the use of percentages (rather than mole fractions) in the numerator of Eq. (13). The calculations for CO2 and C2H4 are fully analogous except: a Those two gases are produced so the final concentration comes first in the subtraction. b In the calculation for CO2 one must further multiply the result by 1.2 to account for dissolved gas (as explained above). The above development has tacitly assumed equilibrium for each species between the phases and also among the dissolved species containing CO2 . The reasoning presented below is intended to justify this. Each molecule of CO2 measured in the gas phase must go through the following steps: synthesis and export by the cells, reaction to H2CO3 , reaction to CO2 and mass transfer from the liquid to the gas phase. The synthesis and transport (taken here as one process) is by far the slowest (hence, rate-determining) step, as is shown below. It is assumed that the CO2 is produced by the cells in the form of H2CO3, HCO–3, or CO2– 3 ; for our purposes, it does not matter which. The chemical reactions between H2CO3 , HCO–3 and CO32– are known to be faster than that between H2CO3 and CO2 [73]. Consider the rate expressions for the forward steps as described in the previous paragraph: 1 Biomass produces carbonate: rH2CO3 = kf [cell mass]

(14)

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2 H2CO3 Æ CO2 + H2O: rCO2 = kCO2 [H2CO3]

(15)

3 CO2(aqueous) Æ CO2(gas): rdeabs = kl a [CO2]

(16)

Since the object of the exercise is to determine how quickly equilibrium is established, we are only concerned with the time before the reverse reactions. Here the first two values of r are the rates of formation (in, for instance, millimoles of species per liter per hour). The quantity rdeabs is the rate of deabsorption of CO2 , in the same units as the other two rates. The brackets indicate liquid-phase concentration and the coefficients of the bracketed terms are the respective rate constants, which will be described below. If any of the rate constants is much lower than the others, that step is the slowest, therefore ratedetermining, step. As will be demonstrated below, this is indeed the case. The value of kCO2 is 20 s–1 (or 7200 h–1) at room temperature [73]. The value of kla, the interphase mass transfer coefficient, for our system must be estimated based on literature reports of mass transfer rates in relatively similar systems. Although the estimate of kl a obtained below is highly uncertain, it is orders of magnitude less than kCO2 but very much larger (hence, less ratedetermining) than kf. 2.2 Absence of Significant Mass Transfer Resistance

The mass transfer resistance at a liquid-vapor interface results from two resistances, the liquid boundary layer and the gas boundary layer. In conditions involving water and sparingly soluble gases, such as occurs here, the liquid-phase resistance is almost always predominant [71]. For this reason, equation (16) involves only kl , the mass transfer coefficient across the liquid boundary, and a, which is the gas bubble surface area per unit volume of liquid. Often, as here, those factors cannot be estimated individually, so kla is treated as a single parameter. The applicability of the O’Connor–Dobbins correlation [73] can be verified using data from Kobayashi et al. [50] They report placing 30-ml volumes of medium in 100-ml Erlenmeyer flasks on a reciprocal shaker at 100 strokes/min. They do not report the stroke length, but a 1-inch stroke is a reasonable guess. Under such conditions, the average liquid depth was roughly 1.3 cm, velocity was 8.5 cm/s, and D = 2.4 ¥ 10–5 cm2/s (they were measuring Kla for O2). The O’Connor–Dobbins correlation gives kla = 19.5 h–1. Kobayashi et al. [50] measured Kl a (capital K because they measured the global mass transfer coefficient) to be 17.5 h–1, certainly close enough to the correlation to justify the latter’s use as a reasonable approximation. The results of stoppered culture tests that we conducted using A. annua suspension cell cultures in shake flasks are used to evaluate kf , the formation rate constant for dissolved carbonate from biomass. Our experimental values ran–1 ged from 0.1 to 0.25 millimoles CO2 g–1 dw h of biomass. Since the biomass quan-

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tity never exceeded 0.8 g in any of the tests, the production rate never exceeded 0.2 millimoles h–1 (the arithmetic product of the maximum productivity and the maximum biomass). This rate was enough to change the headspace gas composition to 1.25% (the approximate uncertainty of measurement of gasphase composition) per hour. Thus, in the time it took for the composition to change by an amount barely detectable, nearly 20 time constants of mass transfer occurred. During each time constant, the difference between equilibrium and actual concentration diminished by a factor of 1/e (e being the base of the natural logarithms, approximately 2.72). The lag due to mass transfer resistance was very much less than the uncertainty in the measurement. Similar calculations show that the O2 and C2H4 concentrations were sufficiently close to equilibrium to warrant neglecting any mass transfer resistance. In our culture experiments in which headspace gas concentrations were kept constant by the flow of defined gas mixtures of O2 , CO2 and C2H4 through the headspace of the cultures (gas feed tests) [27–30], the same processes of H2CO3 production, reaction to CO2 and deabsorption of CO2 (governed by Eqs. 14–16) occur. The rate constants kCO2 and kl a should be identical in the gas feed tests to those in the stoppered culture tests that are discussed below. The value of kf probably varies as a function of the dissolved gas composition. However, its value would have to be at least an order of magnitude greater for the carbonate formation rate not to be the rate-determining step. Since this is implausible, it is quite unlikely that dissolved CO2 concentration in the culture medium of the gas feed tests would be any different than that in equilibrium with the headspace gas. Again, the same reasoning would apply to the O2 and C2H4 concentrations. 2.3 Calculation of Specific Oxygen Usage Rate Throughout a Test

The headspace gas concentrations of the stoppered cultures were measured periodically until they stopped changing significantly. In particular, the consumption of O2 was followed as a marker of cellular activity. Oxygen consumption, rather than CO2 production, was chosen as an indicator of metabolic activity, for three reasons. First, the total amount of O2 in solution was much less than the amount in the headspace so any uncertainty in the former quantity was an insignificant fraction of the total amount of O2 . Second, production of CO2 can occur due to both aerobic and anaerobic metabolism. The latter process, noticeable under reduced O2 concentrations (the Pasteur effect), results in less growth but more sugar usage and much more CO2 production than does the aerobic process [38]. Use of CO2 output as an activity indicator could exaggerate the activity occurring at low oxygen concentration. Third, because of relative concentration differences, the analysis of O2 concentration was more accurate than that of CO2 . The gradual reduction in cellular activity, culminating in culture death a few days after cultures were stoppered, was clearly the result (directly or indirectly) of changes in dissolved gas concentrations. Otherwise, the cells would have continued actively metabolizing as long as did the conventional unstoppered cultures. The conventional cultures, capped with permeable foam and foil, con-

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tinued growing at least 13 days [74]. The simplest explanation of declining activity is the declining O2 concentration. The following series of steps was performed for each stoppered culture test in order to obtain a plot of specific oxygen usage as a function of oxygen concentration. The O2 concentration at each time was converted to total millimoles NO2 of O2 in the headspace. In each test, a suspension culture of A. annua was flushed with a gas mixture of known composition, then sealed and placed on a rotary shaker. The gas headspace was sampled at several hour intervals; the measured concentrations from all the tests were used to calculate the O2 consumption and CO2 production rates. 2.4 Effect of Headspace Composition on Growth and Production of Artemisinin

Four types of stoppered culture tests were performed as follows: a b c d

New culture from 21-day-old inoculum, flushed with air, shaken at 95 rpm Same as ‘a’ except flushed with high CO2 , high O2 gas mixture Same as ‘a’ except tests were started with 7- or 8-day-old cultures Same as ‘a’ except inoculum was from a 10-day-old culture

The initial specific rates of O2 consumption and CO2 production are calculated and interpreted, and the effect of diminishing O2 concentration on its specific consumption rate is investigated. For each test type, the mean and standard deviation of the O2 and CO2 specific rates are calculated and tabulated in Table 1. The four types of stoppered culture tests were compared using two-sample t tests on these quantities to determine whether the particular conditions affected the cell metabolism [74]. If either initial rate showed a statistically significant (95% confidence level) difference between the two test types being compared, then we concluded that the conditions affected metabolism and would be likely to affect bioreactor performance. Table 2 shows the comparisons made and the probability that the difference between the sets of data would occur if the sets were actually from the same population. The results and their interpretations follow: 1 a vs. b (effect of flushing with air vs. flushing with a high CO2 , high O2 gas mixture): Table 1. Summary of gas exchange data from stoppered culture tests using A. annua. Mean initial specific rates of O2 usage and CO2 production (and their standard deviations) in –1 h–1 mmol gdw

Test type a

Number

Mean O2

SD O2

Mean CO2

SD CO2

a b c d

7 4 4 3

0.153 0.232 0.166 0.154

0.079 0.048 0.023 0.056

0.152 0.072 0.222 0.240

0.045 0.116 0.007 0.017

a

Test types are explained in Sect. 2.4.

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Table 2. Summary of gas exchange data from stoppered culture tests using A. annua. Two

sample t test results Comparison

O2 usage

Equality prob.a

CO2 prod.

Equality prob.a

Conclusionsb

a vs. b a vs. c a vs. d

a

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